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1. 1 t m i 5e 3 E t H t t t 1 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 3 35 40 45 Temperature C Temperature C lt Back Next gt Cancel Click Next to display the van t Hoff and Eyring plots together with a table of thermodynamic constants for the equilibrium and transition state formation In any of the plots right click on a point to exclude the point from the line fitting Ws Thermodynamics Results Create xi m Calculated Thermodynamic Parameters mMm vant Hoff Fiting function vant Hof Terme s Fitting function Eyring Linear 18 5 Parameter Name Parameter Value PER a AH kJ mol AT 495 AS PAK mol 45 037 E TAS kJ mol 1 4 0 37 20 AG kJ mol 48 1E 03 hd Ace AE mel 18 28 ae 3 me 3 3 i n 3 3 5 3 AH ass kJ mol 34 12 eee TE T od A is AS t ass J K mol 18 4 8 5 AT AK B arameters TAS ass kJ mol 53 4 8 Chi 0 01545 LAG ass Ik Limal An 1 2E4032 1KMsK Eyring association 1KsK Eyring dissociation 8 9 9 5 85 x 10 10 5 8 ESL E E 115 Y i 75 E 42 E Hi Y 425 7 43 135 v 65 t t t t t t t t t 1 4 t t t t t t t t t 1 315e 3 325e 3 335e 3 3 45e 3 355e 3 3 65e 3 315e 3 325e 3 3 35e 3 3 45e 3 3 55e 3 3 65e 3 TT 1K AT 1K Parameters Parameters Slope 4133 1 Ms Intercept 21 63 1 MsK P 0 9738 Slope 9033 1 s Intercept 18 84 1 sK Re 0 8764 Help
2. Open New Method Open Close Ctr o Save Save As Ctrl 5 Print Properties lt Recently used files gt Exit 2 3 1 Selecting cycles and sensorgrams During a run the current cycle is displayed by default You can choose which cycle to display in the Cycle selector but the display will revert to the current cycle when a new cycle is started For a completed run choose which cycle to display with the Cycle selector in the toolbar Cycle 13 w Curve Subtracted Fc 3 1 The Curve selector determines which curve in the cycle is current Options in the View menu Section 2 3 4 control which curves are displayed in the sensorgram window 2 3 2 File menu The Open New options for wizard templates and methods create new wizard templates and methods and open existing templates and methods for editing or for starting a run Open opens result files Most result files just display the sensorgrams and tables Files from immobilization and regeneration scouting wizards also display a summary window showing the results of the run see Sections 4 3 2 and 4 4 1 Print prints a hard copy of the results Select the printer to use and check the items you wish to print Biacore T100 Software Handbook 7 version AB Control Software general features Printer Printer epee gm 5 IV File Properties ko c Audit Trail Bere Current Cycle Wizard Template is
3. Regeneration 1 MAMIE KK CE OKK jJ mdr The Report points tab lists the report points in the cycle type definition ordered as far as possible in the order they will appear in the cycle Several injection commands have a predefined set of report points that are added to the list when the command is included in the cycle type Biacore T100 Software Handbook version AB 79 Methods 80 You can add your own report points by filling in the details in the empty row at the bottom of the table A new empty row is added whenever you create a report point Report points are set at defined times in relation to the start or end of injections in the cycle Report points that are set outside the time range for the cycle i e a significant time before the start of the first injection or after the end of the last injection will not be created Note Do not position report points far away from events so that they lose their relevance to the event or so close to an event so that the report point window overlaps the event itself Enter the report point details as follows Name The report point name must be unique within the cycle type Choose a name that reflects the function of the report point Sec Enter the time in seconds between the report point and the event Before Choose whether the report point is to be placed before or After after the event Start of Choose whether the
4. Averaged experimental residuals C Residuals 1 10 T Limit guides Injection start stop Zoom lock e ka and kd increased ck or AA fo orginal p Ka and kd decreased 1200 Biacore T100 Software Handbook 141 version AB Kinetics and affinity analysis To use the tool drag the slider for the modification factor M and observe the behavior of the curve display The original curves which remain unchanged as you drag the slider are shown in black blue curves show the simulation for k and kg multiplied by M while red curves show the simulation for k and kg divided by M If the red reduced rate constants and blue increased rate constants curves clearly diverge from the original curves the fitting is sensitive to changes in the rate constants and the curves probably contain significant kinetic information If on the other hand the divergence is negligible the values of the rate constants do not matter because the binding is fully limited by mass transfer Mass transfer places an upper limit on the rate constants that can be measured on the borderline the fitting is sensitive to a reduction in rate constants but not to an increase Choose the Residuals option in the Compare to frame to examine the effect of varying the modification factor on the difference between the original and modified curves in relation to the experimental residuals The tool
5. Back Finish Cancel 164 Biacore T100 Software Handbook Version AB Thermodynamic analysis Choose whether to use a linear or non linear fitting function for the van t Hoff plot see Section 10 1 1 If you choose non linear fitting a value for AC will be included in the reported parameters Energies of activation E derived from the Eyring plots are also listed for the transition states All thermodynamic parameters are calculated for a temperature of 25 C In any of the plots right click on a point to exclude it from the evaluation Notes Regardless of the setting for the van t Hoff plot the Eyring plots are always fitted to a linear function Calculation of AC by non linear fitting is not valid for transition state data see Section 10 1 2 If you have combined kinetic and steady state affinity data in the thermodynamic evaluation the van t Hoff plot will show all affinity values but the Eyring plots will be empty because the steady state data lacks values for the rate constants Plots of kinetic and affinity constants against temperature show temperature values in C while van t Hoff and Eyring plots use absolute temperature values K Click on Finish to finalize the thermodynamic analysis When assessing the validity of thermodynamic constants reported by this analysis procedure pay particular attention to the kinetic analysis at different temperatures With complex interactions involving macromolecules
6. Window 5 7 fs Position the report point ig seconds after Cycle start 7 IV Calculate response relative to report point Help Enter the id for the report point maximum 30 characters in the Id field The report point id must be unique within an evaluation session Enter a value between 1 and 35 for the Window The response value for the report point is the average response over the window with the report time at the center of the window A window of 5 seconds is recommended for most purposes Biacore T100 Software Handbook Version AB Evaluation software general features Use the settings in the Position the report point field to define where the report point will be placed Report points are placed a specified number of seconds before or after the beginning and end of injections or the beginning or end of the cycle Note Do not position report points far away from events so that they lose their relevance to the event or so close to an event so that the report point window overlaps the event itself Check Calculate response relative to report point and select the required report point if you want to calculate the responses at the custom report point relative to another report point If the box is not checked the response will be calculated relative to the closest preceding baseline report point Check the assay step purpose s for which you want the report point to apply in the Assay Step P
7. pum 20 30 t t t t t t 1 5 0 5 10 15 20 25 30 Cycle number 16 658 Medium 9 393 Medium 35 776 Low T Ranking results are independent for each plot window Note Editing the definition of a plot does not affect ranking boundaries If you for example change the y axis parameter of a ranked plot from relative response to absolute response you will need to revise the placing of the ranking boundaries if the ranking is to remain meaningful 118 Biacore T100 Software Handbook Version AB Data presentation tools 7 3 Report point table Report points are automatically created for all wizard and method based runs placed at strategic positions in relation to injections See Section 6 5 for details of how to add custom report points 7 3 1 Displaying the report point table The report point table lists numerical values for all report points in the current result set Choose Evaluation Add Report Point Table to create a report point table window You can only add one report point table window to each evaluation session the table is updated automatically if you add custom report points or apply solvent correction oix Cycle Fc Time s Window s AbsResp RU Slope RU s LRSD Baseline RelResp RU Report Poir a 1 5 389047 0 6139 0 3279 0 02705 No 15 4 stability 2 140 5 389542 0 4881 0 256 0
8. Biacore T100 Software Handbook Version AB Application wizards Te Immobilization Results Ioj xl Chip CM5 Response Response Flow cell Procedure Method Ligand Bound RU Final RU Target Reached 2 Target level Amine anti beta2micro 1231 8 1244 9 Blank Amine 300 5 Time and flow Amine ant beta2micro 721 1 708 8 The summary lists the procedure and method the name of the ligand and whether the target was reached with Aim for immobilized level Two response values are reported one directly after the ligand immobilization and one after the deactivation injection The difference between these values is an indication of the amount of non covalently bound ligand that is washed from the surface by the deactivation injection Note that the Response bound value does not include the contribution from activation by EDC NHS For low ligand levels this value usually gives a better indication of the amount of ligand immobilized RU 60000 T 55000 50000 45000 40000 35000 7 Immobilized Response Response Response final bound 30000 at tg tt 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Time s Result files from immobilization can also be opened in the Evaluation Software if you want to prepare other sensorgram displays or plots see Chapter 7 Biacore T100 Software Handbook 39 version AB Application wizards 4 4 Assay development wizards 40 4 4 1 Regeneration scout
9. however including control samples is generally recommended as an aid in assessing the performance of the assay Check the Repeat control samples every box and enter a number of cycles to repeat the control samples at intervals during the run The controls will then be run at the beginning of the assay and at the specified interval If this box is not checked the control samples will be run once at the beginning of the assay Since control samples are presented in the evaluation software as a trend plot see Section 8 2 2 running repeated controls throughout the assay is recommended Control samples are specified in terms of sample ID and expected concentration The expected concentrations should lie within the range covered by the calibration curve Biacore T100 Software Handbook Version AB Application wizards 6 Samples xi r Sample table Sample id Dilution factor Sample 1 Sample 2 5 Sample 3 Help Import lt Back Nest gt Close Enter the details of the samples to be analyzed Each sample is defined by a sample ID and a dilution factor the dilution factor is used during evaluation to calculate the measured concentration in the original undiluted sample For undiluted samples dilution factor 1 the Dilution factor column may be left blank Samples will be analyzed in the order entered To analyze replicate samples enter same sample on multiple rows Click I
10. lt AssayStep name Startup gt lt DataTable row 1 gt Data cmd Sample 1 grp fld Solution val sample 1 2 gt Data cmd Sample 1 grp fld Conc1 val 0 gt Data cmd Sample 1 grp fld Conc2 val 125 gt Data cmd Sample 1 grp fld MW1 val 200 gt Data cmd Sample 1 grp fld MW2 val 10000 gt lt DataTable gt lt AssayStep gt lt AssayStep name Sample gt lt DataTable row 1 gt Data cmd Sample 1 grp fid Solution val sample 1 2 gt Data cmdz Sample 1 grp fld Conc1 val 0 gt Data cmd Sample 1 grp fld Conc2 val 0 gt Data cmdz Sample 1 grp fld MW1 val 200 gt Data cmd Sample 1 grp fldz MW2 val 10000 gt z DataTable DataTable row 2 gt Data cmd Sample 1 grp fld Solution val sample 1 2 gt Data cmd Sample 1 grp fldz Conc1 val 0 gt lt Data cmd Sample 1 grp fld Conc2 val 195 gt ml Part of an XML import file from a method for heterogeneous analyte kinetics displayed in an XML compatible editor Rack positions import The Menu Import Positions function in the Rack Positions dialog Section 4 2 5 imports rack position data from an external file such as one from a laboratory robot used to prepare sample microplates The external file is first processed by the import program as specified in Files Preferences Output from this program must be tab separated t
11. Flow path h Help lt Back Next gt Close You can only choose a flow path that is consistent with the Detection setting for the method see Section 5 4 5 9 2 Variables In this step you assign values to variables that are to be defined at run time typically a sample table see Section 5 7 Each row in the variables table corresponds to a cycle in the run A new empty row Biacore T100 Software Handbook Version AB Methods marked with an asterisk is created automatically at the bottom of the table as soon as data is entered Columns in the table correspond to variables for the cycle type used in the assay step and are grouped according to commands in the cycle type definition Te BufferChange Yariables xj 1 Select assay step m2 Enter values for Assay Step Buffer A Sample 1 Solution Conc uM Dilution BatchNo Sample 1 15 1 44387 Sample 2 32 1 ERs Use the right mouse button in the variables table to access functions for copying and pasting cell contents and for inserting and removing rows The columns in the table are listed in the order they are defined in the method see Section 5 6 2 Click Import to import the variables table from an external file See Appendix A for details of the import format Depending on how the method is defined there may be variable tables for several assay steps Data must be entered in all tables before you
12. Note Verification only checks the consistency and completeness of the method It does not in any way verify that the method is suitable for the intended purpose Each aspect of Method Builder is described in detail in the following sections 5 3 Method overview T BufferChange General settings General Settings Concentration urit uM Data collection rate 1Hz Startup Sample analysis 3 times as entered Sample compartment temperature 25 C EH Detection Muli Cycle Types Buffer A Sample Sample analysis 1 time as entered Settings for assay step Startup Sample amp Seh Temperature 25 C En Controls buffer A Buffer A Varfication fontrot Semple Sample analysis 1 tine as entered Before every 10 cycles t DMSO buffer A Settings for cycle type Sample analysis Solvent Correction Solvent correction 1 time as entered Before every 15 cycles I Regenerat ge E Sample 1 varies by cycle varies by cycle varies Type High performance Solution varies by cycle Contact time s varies by cycle Conkrots better n Flow rate ulmin 30 Control Sample Sample analysis 1 time as entered Before every 10 cycles Flow path 1 2 3 4 Sample analysis 1time as entered 8 Dissociation time s varies by cycle DMSO buffer B Predip No Solvent Correction Solvent correction 1 time as entered Before every 15 cycles Mix No Extra wash No Stabiliza
13. difference between AbsResp and baseline in RU N A if no baseline has been set Report point Report point id CycleType Identifiers for the cycles set explicitly in methods see Chapter 5 and automatically in wizards AssayStep AssayStep Purpose Keywords One column is created for each keyword in the data Sorting and filtering tbe report point table The report point table can be sorted by any column in ascending or descending order for any column Click in the header for a column to Biacore T100 Software Handbook Version AB Data presentation tools sort the table by that column Each click in the same header toggles the sort order PP Cycle Fc Time s Window a AbsResp eur Filter row e m A E ee A H 6 1688 1508 3805E404 Header row The second row in the report point table contains a filter setting for each column All values will be included if the filter setting is blank To apply a filter click on the setting and select a value from the list The value will be shown in the filter setting and only rows in the table that contain the value in the selected column will be displayed You can apply multiple filters to the table at the same time To remove a filter choose All from the list of column values in the filter setting Copying report point table contents To copy the contents of the report point table select cells by dragging with the mouse and press Ctrl C or c
14. kd2 and second analytes s kd2 KD1 Equilibrium dissociation constant for kd1 kal KD2 the first and second analytes M kd2 ka2 Rmax1 Analyte binding capacity of the Rmax2 rcf surface for the first analyte RU mw1 mw2 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis Rmax2 Analyte binding capacity of the Rmax2 surface for the second analyte RU Concl Analyte concentration M Conc1 Conc2 Conc2 tcl Flow rate independent component of tc1 tc2 the mass transfer constant for the first tc2 and second analytes Flow Flow rate pl min f kt1 Mass transfer constants for the first tc1 13 kt2 and second analytes tc2 13 RI Bulk refractive index contribution in RI the sample 9 3 4 Kinetics Heterogeneous Ligand This model describes an interaction between one analyte and two independent ligands The binding curve obtained is simply the sum of the two independent reactions Unlike the case of heterogeneous analyte the relative amounts of the two ligands does not have to be known in advance Heterogeneous ligand situations frequently arise in practice through heterogeneous immobilization of ligand e g amine coupling of proteins where the ligand has multiple attachment points as well as through heterogeneity in the ligand preparation itself In cases where the heterogeneous ligand model is found to give the best fit to the observed sensor
15. or new parameter settings and click Fit To remove a fit from the evaluation item select the fit in the list of current fits and click Delete Click Finish to complete the evaluation and place the item in the evaluation explorer panel You can click Back to review the choice of data for the evaluation however if you make any changes to the data e g remove additional sections from a curve or exclude a curve from the set all current fits will be deleted Current fits are also deleted if you switch between kinetics and affinity evaluation Kinetics results When the fit is completed the results for kinetics are displayed as fitted curves overlaid in black on the experimental data with details in the table below the curves EB kinetics Affinity Fit Kinetics Create xj Add Fit Current Fits Modet 1 1 Binding zi Description o Parameters Fit Delete Check Kinetic Data RU 804 Response S 5 200 o 200 400 600 800 1000 1200 1400 Time s TEET Report Residuals Parameters ka 1 Ms kd 1 5 KD M Rmax RU Conc M tc Flow ul min kt RU Ms RI RU Chi RUA E 1 743E 5 1 837E 4 1 054E 9 58 9 6 508E 13 0 363 2 5E 08 15 1 605E 14 0 6 2 77E 09 15 1 605E 14 0 5 3 3E 10 15 1 605E 14 0 3 8 33E 09 15 1 605E 14 0 8 9 25E 10 15 1 605E 14 0 6 3 3E 10 15 1 605E 14 0 2 E Biacore T100 Soft
16. same as or close to the value entered for the variable ConcB in the method The calculated Kp value is shown in the panel below the table Biacore T100 Software Handbook Version AB Affinity in solution A Data import and export This appendix describes the functions and data format for data import and export A 1 Exporting data A 1 1 Export functions Data can be exported from both the Control and Evaluation Software in Microsoft Excel or Extended Markup Language XML format The report point table can be exported to a tab separated text file Rack positions may also be exported from the Rack Positions dialog box Export to Excel To export data to an Excel file choose File Export Results to Excel Export from the Control Software creates an Excel spreadsheet file extension xls containing separate worksheets for the file properties and report point table The audit trail is also exported to a separate worksheet if it is present when the GxP module is installed see the separate Biacore T100 GxP Handbook Export from the Evaluation software an Excel spreadsheet file extension xls containing separate worksheets for the file properties and for tabulated data for all evaluation items where appropriate i e plot data and evaluation results The worksheets for each item are identified with the item name For plots only the columns shown in the plot window table are exported Data from sensorgram items is not exported The au
17. the run until the analysis temperature is stable at the new value You can choose to ignore temperature instability but the response will drift as the temperature stabilizes The absolute response decreases by about 150 RU for a 1 C increase in temperature Sample compartment temperature This is the temperature in the sample compartment Equilibration of the sample compartment to a new temperature will start when the run is started The system will not wait for a stable sample compartment temperature at the beginning of the run samples equilibrate to the analysis temperature during passage through the IFC so that the sample compartment temperature is not critical for the measured SPR response Biacore T100 Software Handbook 27 version AB Application wizards 28 4 2 5 Rack positions This dialog box shows where samples and reagents are placed in the microplate and rack Positions are color coded according to sample and reagent categories you can change the color coding in the Automatic positioning dialog accessed through the Menu button Tis Method Builder Rack Positions ni xj Reagent Rack 2 position Sample 1Conci nM Sample 1 Cor o 0 e je RIAS 88 sample 1 2 Sample 0 781 RI A4 88 sample 1 2 Sample 0 3125 R1A5 88 sample 1 2 Sample In o RI A6 88 sample 1 2 Sample 0 125 RIA 88 sample 1 2 Sample 0 500 Ri Ag 88 sample 1 2 Sample
18. 0 125 R1 A9 88 sample 1 2 Sample jo 0 RI Alo 88 sample 1 2 Sample 0 125 RIAI 88 sample 1 2 Sample 1195 aas RI A12 88 sample 1 2 Sample 7 81 125 WUyy 000 Ri Bt 88 sample 1 2 Sample 3125 125 100000000 Ri B2 88 sample 1 2 Sample 0 0 10 R163 88 sample 1 2 Sample 125 125 6 00000000 Ri B 88 sample 1 2 Sample 500 125 00000000 R1 B5 88 sample 1 2 Sample 0 125 id e Q Q Q Q Q Q R1 B6 88 sample 1 2 Sample o 0 OOOOQOOOQO R1 B7 88 sample 1 2 Sample 500 0 R1 CI i 244 sample 1 2 Startup 0 125 00000000 R2 Al i 1334 Reg solution Regeneration OOOOOOOO O0000000 00000000 69000000 OO60OOOO0 A B c D E F 6 H You can change the reagent rack and microplate types in the pull down lists above the respective illustrations If you do so all positions in the affected rack or plate will be cleared and must be reassigned either manually or automatically Positions are described by tool tips place the cursor on the position for a couple of seconds to display the tool tip Empty positions show the position capacity and dead volume Used positions show in addition the content name and the volume that will be used You can change sample and reagent positions manually in two ways e Click on a sample or reagent in the sample plate and rack illustration and drag it to a new empty position You cannot drag to a position that does not have sufficient capacity for the required volume of sample or reagent e Type a position
19. 1 Binding zi Parameters amp X 800 1000 1200 1400 s lt Back Finish Cancel Select the model from the pull down list Available models are described in Section 9 3 Click Parameters if you want to change the starting values or scope of any of the parameters see Section 9 2 2 for details then click Fit to perform the fitting During the fitting procedure the fitted curves are shown in black overlaid on the experimental data Fitting progress is indicated in the sensorgram window by display of the iteration number the current chi squared value and the relative change in the parameter that was changed most from the previous iteration You can use the Abort or Accept buttons to cancel the fitting or accept the fitting after the current iteration You may want to cancel the fitting if it is clear that a fit cannot be found or to accept the fitting if the chi squared value and or maximum relative change indicate that an acceptable fit has been achieved Clicking on Biacore T100 Software Handbook Version AB Kinetics and affinity analysis Accept will stop the fitting at the end of the current iteration which may take a few moments to complete You can enter a short description for the fit in the Description box This can be useful for example to distinguish different fits for thermodynamic analysis Section 10 2 To perform additional fits on the same data choose a new model
20. Add Fit Current Fits Model Steady State Affinity zi Description Parameters Fit Delete RU amp 0 4 m ssi 40 4 loe 1 p ad er E Pa 204 x x E 150 8 Concentration Report Parameters KD M Rmax RU RI RU Chi RUZ 1 277E 8 79 3 0 6 0 191 Help lt Back Finish l Cancel i 136 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis The detailed results are presented on two tabs e Report shows selected parameters and calculated values The contents of the Report tab are defined in the model Global parameters are listed on a single row at the top of the table and local parameters are listed on one row for each curve e Parameters shows the values for all parameters in the fitting equations 9 1 3 Multiple ligand densities Analysis of the same analyte concentration series over multiple ligand densities can provide more robust fitting than a single ligand density The kinetic and steady state affinity fitting functions support simultaneous analysis of up to five sets of data with independent values for Rmax returning a single set of rate constants for the whole combined data set Analyses over multiple ligand densities may be performed in separate runs that are combined with the Append file function but should preferably be performed on multiple flow cells in the same run to ensur
21. For up to 4 days Flushes the IFC and flow cells with buffer at a low Flow rate The software will be shut down as soon as the standby procedure has been started NN 2 3 3 Edit menu Options in the Edit menu allow you to add edit and delete report points Report points are created automatically and are used in various evaluation contexts You should in general avoid editing or deleting report points that are created automatically Editing operations for report points in the Control Software may be applied to single curves or to all curves in the current cycle Note that editing operations are not applied to multiple cycles Report points created in the Control Software cannot be edited in the Evaluation Software The Evaluation Software offers functions for creating and editing custom report points that can be applied to all cycles in the run in a single operation This is usually preferable to adding report points in the Control Software Biacore T100 Software Handbook 9 version AB Control Software general features view Chip Properties Title Scale Adjust Scale Unzoom Ctrl v Reference Line Baseline F8 F9 Show Only Current Curve Show Curves of Same Type v Show All Curves Event Log Wizard Template Notebook 2 3 4 View menu Chip Properties opens a dialog box that displays the properties of the currently docked sensor chip The Ligand column is empty for flow cells that
22. Handbook Version AB Data presentation tools iix Zi Curve Name Fc 2 1 v 4 Assay Step Purpose Overlay v v 4 Cycle lt Overlay gt v v Tes v RU Sensorgram Zoom Lock 1700 4 B 1650 4 1600 4 1550 4 1500 4 v Caption 8 Cut 1450 4 Scale x Copy Graph 1400 7 Export Curves Gridlines 1350 4 Legend Choose Undo Cut from the right click menu to restore the removed data in the display 7 1 3 Sensorgram adjustment Choose Sensorgram adjustment under the Tools button for options for aligning and adjusting the sensorgram display For curve alignment sensorgrams which do not include the chosen reference point for alignment will not be shown Biacore T100 Software Handbook 111 version AB Data presentation tools 112 I Adjust Sensorgram lal xl Adjustment C Off is Report Point time Q z Injection Event time 0 Sample 1 start m Y Adjustment I C Off Report Point response 0 baseline C Injection Event response 0 z Report Point response 100 z Injection Event response 100 z M Blank Subtraction A v Enable Blank Subtraction Cycle 11 Curve Fc 4 3 Help DK Cancel X adjustment Choose to set the zero time point to either a report point or an injection event If this setting is Off the zero time point will be at the beginning of the cycle Y adjust
23. RU sample injection Response RH RI2 baseline y WY Time Check the Drift box and enter an expression describing the drift most commonly a linear function of time to account for baseline drift Biacore T100 Software Handbook Version AB Kinetics and affinity analysis 2 Enter the reaction scheme in the Reaction panel using the pull down list for each reactant Enter parameter names for the forward and backward rate constants for each line in the reaction scheme The terms k forward and k backward apply to the reaction as entered in the scheme reading from left to right You can also enter mathematical expressions or constant values for the rate constants 3 Click the Parameters tab and define the parameters used in the reaction scheme Click Add to add a new parameter and define the parameter properties in the dialog x Name ka2 Default Type Fit global m Initial Value Value 1e5 C Attach to z Allow negative value Description Second association rate constant Help DK Cancel Choose a default type for the parameter Fit global Fit local or Constant For the Initial value enter a numerical value or select a value expression from the pull down list The expression represent functions evaluated within the current data set e g Ymax is the maximum y value in the data set Alternatively choose Attach to and select a parame
24. Rows for which a match cannot be found are not imported Any rows in the Rack positions table which do not have a matching row in the import file are left without a Position specification and must be placed in the microplate or reagent rack before the run can be started Details of the required import file format can be investigated further by examining a file created with the Menu Export Positions command A Rack1 96 Well Microplate Rack2 Reagent Rack 2 Position Volume ul Content Type Sample 1 Conc pj Sample 1 MW Da R1 A1 88 a Sample 1 R1 A2 88 b Sample 0 R1 A3 88 b Sample 1 R1 Ad 88 b Sample 1 R1 A5 88 b Sample 2 R1 AB 88 b Sample 3 R1A7 88 b Sample 4 R1 A8 88 b Sample 5 R1 A9 88 a Sample 0 R1 A10 88 a Sample 1 R1 A11 88 a Sample 2 R1 A12 88 a Sample 3 R1 B1 88 a Sample 4 R1 B2 88 a Sample 5 Example of an exported file for rack positions opened in Microsoft Excel Biacore T100 Software Handbook Version AB Affinity in solution A 2 2 Evaluation Software The Evaluation Software supports import of model definitions for kinetics and affinity evaluation Model files for import should be obtained from Biacore or created by exporting models from another installation Biacore T100 Software Handbook 175 version AB Affinity in solution 176 Biacore T100 Software Handbook Version AB Error Reference source not found Index 1 1 1 binding 145 A aborting fitting procedure 134 accep
25. affinity are one cycle with a Sample injection in an assay step with purpose Sample with a sample concentration in the keyword Conc If the concentration is not given in molar units the keyword MW must also be included with a value for the molecular weight For method based runs the method must be correctly constructed as described in Section 5 10 2 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command name cannot be edited in the keyword table Refer to Chapter 5 for details of how to construct methods in Method Builder The recommended minimum conditions for kinetic and affinity evaluation are e aconcentration series of analyte with at least five non zero concentrations and one zero concentration e duplicate determinations for at least one non zero concentration These conditions are recommended but not mandatory in the Kinetics Affinity wizard Biacore T100 Software Handbook 129 version AB Kinetics and affinity analysis 9 1 2 Evaluating kinetics and affinity To start a kinetics or affinity evaluation click the Kinetics Affinity button on the toolbar or choose Add Kinetics Affinity from the Evaluation menu 130 The first dialog presents the concentration series available in the current result set and allows you to choose the curves included in the evaluation EX kinetics affinity Select Curves Create xi Curves Sample ss m T
26. as an expression defining response as a function of time t To create an equation model choose New in the kinetics models dialog and create a new model without using the currently selected model as a template 158 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis ES Edit Model Dissociation RO exp kd t t0 Offse Parameters and report parameters are defined in the same way as for kinetic models Biacore T100 Software Handbook 159 version AB Kinetics and affinity analysis 160 9 4 3 Models for steady state affinity Models for steady state affinity evaluation are entered as an expression defining Reg as a function of concentration Conc EB Edit Model x Model Name Two site affinity Description ee state affinity with two ligand sited Formula Cone Rmax1 Conc KD1 Conc Rmax2 Conc KD 2 Independent variable Conc Parameters Allow Type negative Description value Fit global XMaxj2 Fit local Max 2 Fit global XMax 2 Fitlocal YMaxj2 Parameters and report parameters are defined in the same way as for kinetic models Note Beware of trying to define and use complex models for steady state affinity Because of the relatively few points available for fitting to steady state affinity models typically about 5 concentrations in duplicate complex models tend to give unstable fitting b
27. assay but not required by an application specific evaluation procedure an example might be the sample batch number Evaluation variables may only be defined for Sample and General commands Variables are set in the list at the right of the command panel For method variables check a parameter to set it as variable For evaluation variables choose the purpose of the assay to display an appropriate list of predefined variables and check the variables you want to use Click Add to set up user defined variables Note For specific assay purposes you should generally check all suggested variables If you leave some variables unchecked the assay specific evaluation may not work Method Variables Evaluation Variables Method Variables Evaluation Variables Set property as variable Predefined variables Solution Name Value type Contact time s Conc Numeric Dissociation time s Dilution Numeric O Flow rate ul min User defined variables Name Value type BatchNo Text Add Delete Evaluation purpose Predefined evaluation variables for different assay purposes are described in the table below see also Section 5 10 Assay purpose General Kinetics Affinity Thermodynamics Conc Analyte concentration MW Analyte molecular weight required for molecular weight adjustment of report points and for kinetic evaluation when the concentration is entered in weight based units Biacore T10
28. at the left and a table of x and y values at the right Tool tips identify the data points place the cursor on a point for a couple of seconds to display the tool tip 114 Biacore T100 Software Handbook Version AB Data presentation tools BB Binding levels 4 Curve Name Fc 2 hi v 4 Assay Step Purpose Overlay z 44 Cycle number Overlay v RU Binding levels Zoom Lock e e E E t t A 8 14 Fc 2 Sample b2u 5 18 4 f o 2 5 z t Right click on the plot item in the evaluation explorer and choose Rename to change the plot name 7 2 1 Selector functions The selector bar at the top of the window controls which points will be displayed E Curve Name Fc 4 v v 4 Assay Step Purpose lt Overlay gt gt v E Cycle Overlay F v e Curve name lists the type of sensorgram from which the points are taken active reference reference subtracted and solvent corrected where applicable e Assay Step Purpose filters the points according to the assay step purpose e The third selector lists the variable values represented on the x axis This option is not available for plots of report point against report point Selection operates in the same way as in the sensorgram window Section Error Reference source not found Other general display functions are described in Section 6 3 Biacore T100 Software Handbook 115 version AB Dat
29. baseline Choose the optimum buffer pH on the basis of the binding behaviour at pH suitable for immobilization the ligand binds rapidly to the surface during the injection and dissociates completely after the end of the injection The optimum pH is generally the highest value that gives sufficient ligand binding not necessarily the value that gives the highest ligand binding Beware of conditions that give irregular sensorgrams with incomplete dissociation this behavior often indicates aggregation or denaturation of the ligand 4 3 2 Immobilization The immobilization wizard supports immobilization of ligand in any combination of the four flow cells in one run Immobilization in each flow cell is performed independently in a separate cycle so that different ligands and or immobilization conditions can be used in the different flow cells See the Biacore Sensor Surface Handbook for more information about ligand immobilization Biacore T100 Software Handbook Version AB Application wizards 1 Immobilization setup Te Immobilization Immobilization Setup xj 7 Chip type CM5 hd Flow cell 1 Ie El Immobilize flow cell 1 Method L8 Amine C Aim for immobilized level Specify contact time and flow rate Blank immobilization Flow cell 2 7 Immobilize flow cell 2 Method R Amine C Aim for immobilized level Ligand ligand 1 Dilute ligand Specify contact time and flow rate Contact tim
30. directly in the Position column in the table or copy Ctrl C a position from one row and paste it Ctrl V in another Positions can also be reorganized using the Automatic positioning dialog see below Biacore T100 Software Handbook Version AB Menu Y Eject Hack Clear Positions Default Positions Automatic Positioning Save Wizard Template Save Wizard Template As Automatic Position Import Import Positions Export Positions Print Rack Positions Print Wizard Template version AB Application wizards Menu functions Use the Menu button to access additional functions for rack positioning Clear Positions This option clears the entries in the Positions column for the selected rack or plate Clear Positions xj This will clear all sample positions in the selected racks v Reagent Rack IV Microplate Help Cancel Positions that are cleared must be reassigned before the run can be started Default Positions This option restores all entries to default positioning The default positioning is determined from the type and volume of solution in combination with the currently selected rack type Choosing Default Positions overrides any changes that have been made in the rack positions even if the changed positions have been saved in the wizard template Automatic Positioning This option controls the way samples and reagents are positioned automatical
31. display allows the simulated difference curves to be compared to the experimental residuals or to residuals averaged over a moving time window The latter option smooths the experimental residual display making it easier to observe the general shape of the residual curves Movable horizontal Limit guides can be displayed to mark the extent of the residual variation and aid visual interpretation Note that the limit guides do not in any way imply acceptance limits 00 0 100 200 300 40 50 80 700 800 900 ao o 100 200 300 400 500 600 700 800 900 Time Time Kinetic data check comparison to residuals left and averaged residuals right Note To use the Check Kinetic Data tool on a finished kinetic evaluation item open the item for editing and step forward to the last stage of the kinetic evaluation Standard error or T value The significance of parameter values is indicated by the standard error SE or T value listed on the Parameters tab in the fitting results This is a statistical indication of the significance of a fitted parameter Lower standard error values indicate higher significance if the standard error represents less than 10 of the parameter value the parameter is significant for the experimental data 142 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis For ease of comparison between parameters with widely different absolute values e g k and kg the standard error may be
32. operations in the instrument 1 The specified volume of Deposition solution is transferred in the autosampler to a target position in the sample and reagent rack Target positions are assigned as required in the Rack Positions dialog Section 4 2 5 The deposition solution should be MS compatible and may be used for example to neutralize the recovery conditions which are often acidic or to add trypsin or another protease to the sample for peptide digestion The presence of deposition solution also helps to collect the small recovered volume reliably from the autosampler needle Biacore T100 Software Handbook 75 version AB Methods 2 The Sample solution is injected over the sensor surface with the Contact time and Flow rate as specified The Flow path is fixed so that sample passes through all four flow cells to maximize the amount of analyte that binds to the surface 3 The flow system is washed with the specified Wash solution Distilled water or an MS compatible buffer should be used as washing solution 4 A small volume approximately 2 pl of Recovery solution separated from the surrounding buffer by air segments is injected into the flow cells The flow is stopped for the specified Incubation time while the recovery solution is in contact with the sensor surface to allow the bound analyte to dissociate into the recovery solution 5 The flow direction over the sensor surface is reversed and the recovery solution con
33. optimum immobilization pH 34 P parameter significance 141 parameters for kinetics affinity 134 pausing a manual run 19 pH scouting 32 results 34 plot settings 114 plot window fitted curves 117 table columns 116 plot windows 114 poly His tagged ligands 90 pooling 30 preconcentation 37 preconcentration 32 36 predefined evaluation items 98 predefined evaluation variables 78 predefined immobilization methods 35 predefined methods 87 predefined models 144 predefined plots 99 predip 72 prime 27 printing rack positions 31 printing results 7 printing wizard templates 31 R rack illumination 12 rack positions 28 automatic import 30 Biacore T100 Software Handbook 183 version AB Index 184 default positions 29 export 31 172 import 30 173 rack settings in manual run 16 ranking 118 rate equations 157 158 reactants 154 reagent rack 28 recovering bound analyte 75 recovery solution 76 recurrent assay steps 70 reference line 11 regeneration 24 26 76 manual run 18 regeneration scouting 40 results 42 regeneration solutions viscosity 18 26 41 76 regions 29 regression coefficient 120 regulated environments 2 relative response 120 reliability of Kp values 153 remove selection 133 removing data from sensorgram display 110 replicate assay steps 69 report point identifier 120 report point range 107 report point table 119 export 172 report points 79 1
34. others in the result set To cut data from the sensorgram display drag with the right mouse button to select the data you want to remove and then choose Cut from the right click menu This can be useful to eliminate e g regeneration pulses from the sensorgram display Cutting data affects only the current sensorgram window 7 1 4 Markers You can choose to display markers and or labels for report points and events in the cycle with the Report points and Event markers options respectively under the Tools button Report points are displayed on the curve and event markers on the x axis 47400 mix 4 Curve Name Fc 4 v Assay Step Purpose Overlay r Z Cycle 1 9 v Tools vw RU Sensorgram Furosemide1 10uM I Zoom Lock 47800 7 E y 47700 4 E P f E s B 47600 47500 4 3 7 4 5 i e T t fd 8 8 i E z EI F gi a 5 MJ co baseline 1 capture capture baseline 1 co bindin 47300 2 8 i a 3 8 Da E 2 g 2585 a4 EJ ES E S i E EG S t4 Be SE t a ct sz St e 55 Ss 5 bd S EXE s s a3 c BH amp a d Ba 2 6 e st 8 RS t FE E d pe BR 22 i 2 2 EE g EN E i3 amp Ee of E az u t C Ou 47100 AA A a A ay pai i i a a a mma 0 50 100 150 200 250 300 350 400 Sensorgram display with labels and markers shown for both report points and events Biacore T100 Software Handbook 113 version AB Data presentation tools 7 2 Plot
35. rate constant for the Fitted conformational change s Rmax Analyte binding capacity of the Fitted surface RU Conc Analyte concentration M Provided as input tc Flow rate independent component of Fitted the mass transfer constant f Flow rate pl min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in Fitted the sample Biacore T100 Software Handbook 151 Kinetics and affinity analysis The following values are presented in the report Calculated as ka1 Association rate constant for analyte kal binding M7 s kd1 Dissociation rate constant for analyte kd1 from the complex s t ka2 Forward rate constant for the ka2 conformational change s kd2 Reverse rate constant for the kd2 conformational change s KD Overall equilibrium dissociation kd1 ka1 constant M kd2 kd2 ka2 Rmax Analyte binding capacity of the Rmax surface RU Conc Analyte concentration M Conc tc Flow rate independent component of tc the mass transfer constant Flow Flow rate l min f kt Mass transfer constant tc f1 3 RI Bulk refractive index contribution in RI the sample 9 3 6 Affinity Steady State 1 1 This model calculates the equilibrium dissociation constant Kp for a 1 1 interaction from a plot of steady state binding levels Req against analyte conce
36. relative sizes of the analytes are unknown A1 B A1B A2 B A2B Biacore T100 Software Handbook version AB 147 Kinetics and affinity analysis 148 Model parameters are Obtained from ka1 Association rate constant for the first Fitted ka2 and second analytes M s kd1 Dissociation rate constant for the first Fitted kd2 and second analytes s Concl Concentration of the first and second Provided as input Conc2 analytes M mw1 Molecular weights of the first and Provided as input mw2 second analytes tcl Flow rate independent component of Fitted tc2 the mass transfer constant for the first and second analytes Rmax1 Analyte binding capacity of the Fitted surface for the first analyte RU Rmax2 Analyte binding capacity of the Fitted surface for the second analyte RU rcf Response correction factor allowing Fitted for different refractive index contributions for the two analytes This factor is defined as Rmax1 Rmax2 MW1 MW2 f Flow rate l min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in Fitted the sample The following values are presented in the report Calculated as kal Association rate constant for the first kal ka2 and second analytes M s ka2 kd1 Dissociation rate constant for the first kd1
37. report point is to be placed relative to End of the start or end of the injection Inject Choose the injection to which the report point will be related Window Set the window for the report point calculation The report point will be placed at the center of the window and the reported response will be an average of the response values within the window A window of 5 seconds is adequate for most purposes Baseline Choose whether the report point will be defined as a baseline or not Response values for report points that are not defined as a baseline will be calculated relative to the closest preceding baseline value Biacore T100 Software Handbook Version AB Methods 5 7 Sample and Assay Setup alo x 2 Select where to define variable values for each Assay Step General Settings C Define all variable values at run time C Define all variable values in method Controls buffer B Define some variable values in method and others at run time nse buffer R bd 3 Select where the variable values will be defined Variable values defined in method Variable values defined at runtime Variable Command Variable Contact tine Dissociation time s Samplei Conc Sampel Mw Assay Setup 74 These variable values will be used for all cycles Sample 1 Contact time s Dissociation time s 150 60 This screen determines w
38. run 82 significance of fitted parameters 141 simulate cycle run list 67 Single detection 66 slope 120 solvent correction 103 applying 105 command 77 extrapolation 107 method example 89 principle 105 requirements for evaluation 87 sort keywords 102 sorting report point table 120 Biacore T100 Software Handbook 185 version AB Index 186 specifying variable values 81 SPR technology 1 stabilization period 73 77 stabilization time 26 standard deviation 120 standard error 142 start up cycles 25 in regeneration scouting 40 steady state affinity 133 152 160 stop command in manual run 19 surface performance 43 results 44 system overview 1 system preparations 27 84 T table columns 116 target immobilization level 36 temperature control 1 temperature dependence of kinetics and affinity 164 templates 21 folder 14 21 importing into Method Builder 63 thermodynamic parameters 161 thermodynamics 129 application wizard 57 evaluation 161 requirements for evaluation 86 tool tips 110 Tools menu control software 12 transition state 162 T value 143 two state reaction 151 U unknown samples 123 127 unzoom 10 96 V van t Hoff equation 161 variables 64 77 81 101 entering values at run time 82 entering values in a method 81 verification 65 82 View menu control software 10 viscosity 18 26 41 76 W wait manual run 18 Biacore T100 Software Ha
39. the frequency with which they should be run Check the Repeat control samples every box and enter a number of cycles to repeat the control samples at intervals during the run The controls will then be run at the beginning of the assay and at the specified interval If this box is not checked the control samples will be run once at the beginning of the assay 5 System preparations Check the System preparations options as required see Section 4 2 4 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Evaluation Chapter 9 describes how to evaluate kinetics assays 4 5 4 Thermodynamics The thermodynamics wizard supports kinetic and affinity determinations over a range of temperatures The corresponding evaluation software extracts thermodynamic data from the dependence of rate and affinity constants on temperature see Section 10 1 1 Injection sequence Choose the injection sequence for the assay see Section 4 2 1 The thermodynamics wizard supports capture but not enhancement injections The same injection sequence will be used for all selected assay steps Biacore T100 Software Handbook 57 version AB Application wizards 2 Assay setup x D
40. the method Use the Copy button to make copies of cycle types in the method this can be useful if a method requires a number of similar cycle types with small variations Click Rename to rename a cycle type Enter a description of the cycle type if desired 71 Methods 72 Settings for the currently selected cycle type are configured in the lower part of the work area The settings are divided into Commands and Report Points accessed on the respective tabs 5 6 1 Commands Commands Report Points Capture hd Settings for Sample 1 Type High performance Method Variables Evaluation Variables Insert Command Soluti i jabl Set property as variable I tion s variable Remove From Cycle Solution Contact time Is variable Contact time s Move Up Dissociation time s Dissociation time Is variable LI Flow rate pl min Move Down Flow rate 30 ul min Regen t mew zs ox 1 Predip Dmx wih Fraction T of mix solution TF Stabilization period after mie Onan t Extra wash after injection with oa P o Stabilization period The commands in a cycle definition correspond to different kinds of injection of liquid over the sensor surface To add a new command to the cycle definition choose the command type from the pull down list and click Insert command The command will be inserted with default parameter settings
41. the right hand panel All cycles are shown by default in an overlay plot click the rows in the cycle list outside the Included check box to display specific cycles Clear the checkmark in the Included box to exclude cycles from the correction calculation You must include at least one solvent correction cycle for each curve Sample responses are corrected according to the curve obtained from the nearest preceding correction cycle in the run If there is no preceding correction cycle the nearest following cycle is used Examine the curves for fitting to the experimental points Right click on outlying points to exclude either the single point from the curve fit or the whole correction cycle from the correction process Statistical fitting parameters chi squared are shown for each correction curve in the cycles list Right click on a point or curve in the right panel and choose Show sensorgrams if you want to examine the sensorgrams from solvent correction cycles as an aid in judging the quality of the data In judging the quality of the solvent correction data take note of the y axis scale in the display The curves are automatically scaled to fit the window If the range of solvent correction is small as in the illustration above points may appear to scatter widely from the fitted curve The solvent correction curve is fitted to the experimental points using a second degree equation Beware of applying solvent correction if the correcti
42. the standard error is a mathematical assessment of the significance of a single parameter If the Check Kinetic Data tool indicates that the rate constants are not significant the standard error for the constants may be expected to be high However the converse is not always true a high standard error will not always be reflected in the behavior of the Check Kinetic Data tool Even if parameters with low significance can have a wide range of values without affecting the fit repeated evaluation of the same data set will always return the same value for all parameters Consistency of a value between repeated evaluations is not a test of significance Biacore T100 Software Handbook 143 version AB Kinetics and affinity analysis 9 3 Predefined models A set of predefined models for kinetics and steady state affinity is provided with Biacore T100 Evaluation Software These models are marked in the model selection list see Section 9 1 2 with a red dot and cannot be removed or modified Mass transfer parameters All kinetic models include a term for mass transfer of analyte to the surface If transport is slow compared with binding of analyte to the ligand the transport process will limit the observed binding rate at least partially All models take account of this potential limitation and can extract rate constants from the data provided that mass transfer is not totally limiting see Section 9 2 3 The rate of mass transfer of a
43. then E Then Etse x Regeneration 2 E Else CycleEnd Sample 1 Regeneration 1 Biacore T100 Software Handbook 73 version AB Methods 74 To set up a conditional command 1 Insert an If then command at the appropriate place in the cycle definition Specify the condition This is defined as the outcome of a comparison between a report point value absolute response relative response or slope and a constant or another report point value with an added or subtracted constant value Only report points that have already been set in the cycle definition may be used in the condition Check Use additional condition to combine two conditions using either AND both conditions must be fulfilled or OR fulfillment of one condition is sufficient as a logical operator The available comparison conditions are Greater than and Less than The conditions do not include Equal to since exact equality is an unpredictable condition in view of noise in the SPR response To construct an equality condition combine one Greater than and one Less than condition so that a window of tolerance is created For example the combined condition A greater than B 1 AND A less than B 1 is equivalent to A equals B with a tolerance of 1 Choose the actions to be taken when the condition is met and when it is not met You may choose to execute a command sequence terminate the cycle or the method or introduce a stabilization pe
44. there is a significant possibility that the characteristics of the interaction including the role of mass transport limitations in the observed interaction change with temperature resulting in different fitting quality at different temperatures This may be evident from direct comparison of the kinetic fits but will not be immediately apparent in the thermodynamic analysis Biacore T100 Software Handbook 165 version AB Thermodynamic analysis 166 Biacore T100 Software Handbook Version AB Affinity in solution 11 Affinity in solution Determination of affinity in solution provides an alternative to steady state affinity measurements see Chapter 9 for interactions that take a long time to reach equilibrium or for any other reason are difficult to determine with a direct binding assay In principle the affinity in solution approach uses Biacore to determine the free concentration of one interactant in equilibrium mixtures containing known total interactant concentrations 11 1 Conventions and background 11 1 1 Experimental setup The interactants in affinity in solution determination are denoted A and B A Bz AB Experiments are set up so that a fixed concentration of B is mixed with variable concentrations of A and allowed to reach equilibrium The free concentration of B is then determined by injecting the sample over a ligand that binds B but not the complex AB the interactant A or a derivative thereof is usually suitabl
45. windows Plot windows display report point values plotted against either variables or other report point values in the same cycle Curves can be fitted to points using linear or 4 parameter fitting functions Ranking borders can be added to plot windows to classify response levels into groups such as high medium and low responses To create a plot window click Plot in the toolbar or choose Add plot from the Evaluation menu Enter a name for the plot choose the parameters that define the plot and click Finish m Plot Settings Plot name Binding levels Plot type Report Point vs Variable C Report Point vs Report Point Axis setting Y Axis X Axis Report Point binding 1 Variable Cycle number X Response Type Relative response x Help Finish Cancel Response values may be absolute or relative response or slope or relative response adjusted for molecular weight if the keyword MW is defined Adjustment for molecular weight is performed by dividing the response in RU by the molecular weight in Da Points for which the molecular weight value is zero or missing are omitted from the plot Cycles that do not contain the selected report point will not be represented in the plot Variables may be numerical e g molecular weight or concentration or non numerical e g sample name or assay step purpose The plot will be created with default display settings with a graphical representation
46. windows for options relating to the display The available options vary according to the type of window and also depending on whether you right click on a point a curve or elsewhere in the window The list below describes the functions Labels Displays a label on each point in a plot window showing cycle number flow cell and sample name Labels may overlap and be difficult to read if the points in a plot are closely spaced Caption Displays the name of the evaluation item as a caption in the item window The caption is shown as Empty Plot for sensorgram windows if there is no data to display Show sensorgram Displays the sensorgram relating to a point in the plot This option is only available when you right click on a point the sensorgram is displayed in a separate window that must be closed before you can continue with the evaluation Exclude cycle curve point Excludes data from the evaluation session The data that can be excluded differs according to the type of window Excluding cycles in either sensorgram or plot windows affects all other sensorgram and plot windows correspondingly however application specific evaluation items that have already been created are not affected until they are edited and updated If you have applied solvent correction and then exclude solvent correction cycles the correction remains applied but the excluded cycles will not be available if you edit the solvent correction item Scale Sets t
47. 0 100 120 Chr 13 43 Concentration ng ml A calibration curve is constructed from the cycles in each calibration step If two calibration steps are run in direct succession so that there are no other cycles between the steps they will be treated as a single calibration curve Settings on the Calibration tab define the report point and fitting function that are used to create the calibration curve The settings apply to all calibration curves in the evaluation Choose the settings appropriate for your analysis Flow cell Report point Concentration analysis may be performed without a reference cell since the unknown samples are determined by direct reference to a calibration curve obtained under the same conditions If a reference cell is included in the flow path for the run the reference subtracted curve may however be selected if desired Response levels for concentration analysis relative response are normally taken from a report point shortly after the end of the sample injection to avoid contributions from the bulk refractive index of the sample For analyses based on the rate of binding report point slope a report point early in the sample injection is normally used Response type Choose between relative response and slope Biacore T100 Software Handbook Version AB Concentration analysis Fitting function Choose between linear and 4 parameter functions The 4 parameter function is a gener
48. 0 Software Handbook Version AB Methods Assay purpose Kinetics heterogeneous analyte Conc1 Conc2 Analyte concentrations for the two analytes MW1 MW2 Molecular weight for the two analytes these variables are required even if concentrations are entered in molar units to determine the relative contributions of the two analytes to the observed response Assay purpose Concentration Conc Analyte concentration required for calibration and control samples left blank for unknowns Dilution Dilution factor used for unknown samples to calculate original concentrations Assay purpose Affi nity in solution ConcB calibration Concentration of interactant B used to construct a calibration curve ConcB fix Concentration of interactant B in the sample mixture the concentration of B is kept constant ConcA variable Concentration of interactant A in the sample mixture the concentration of A is varied See Section 11 1 1 for details of how this assay is set up 5 6 3 Report points Commands Report Points Name Sec Before After Start of End of Inject Window Baseline baseline_1 10 Before binding late 1 5 Before stability early 1 10 After regeneration 1 45 After em s eolw I Start of End of End of End of Sample 1 Sample 1 Sample 1 S Yes v 5S No 5 No 5 No
49. 00 119 adjustment for molecular weight 114 editing in control software 9 in regeneration scouting 43 markers 113 Reg against C 133 requirements for evaluation 85 residuals 136 response correction factor 148 right click menus 13 97 Run menu control software 11 run order 56 69 run protocol 31 running wizards 22 S sample and assay setup 81 sample capacity 1 sample command 77 sample compartment illumination 12 Biacore T100 Software Handbook Version AB Error Reference source not found sample compartment temperature 1 27 58 66 sample concentrations 56 sample injection 24 26 sample injections binding analysis 47 sample mixing in concentration analysis 50 sample name in calibration curve 125 sample parameters binding analysis 48 sample response in regeneration scouting 42 samples for calibration curve 51 samples for concentration analysis 53 samples for kinetics affinity 55 scale 10 97 screen regions control software 6 evaluation software 96 screening method example 90 selecting data for thermodynamic evaluation 163 selector bar 7 110 115 Sensor Chip NTA 90 Sensor Chip SA 38 sensorgram adjustment 111 normalizing 112 removing data for display 110 sensorgram adjustment 111 sensorgrams baseline 11 copy graph 13 export curves 13 98 in evaluation 109 printing 8 reference line 11 settings for regeneration scouting 41 settings for Reg 133 setup
50. 03 0 02464 No 21 1 stability 15 140 5 389946 0 1559 0 07433 0 07868 Yes 0 0 baseline 15 54 0 5 39015 9 0 04355 0 01786 0 03123 No 21 4 stability The contents of the report point table cannot be edited The following columns are represented in the report point table File File number This column is only shown when the evaluation session includes more than one file the cycle number is prefixed with a file number Choose File Properties to display the mapping of source files to file numbers Cycle Cycle number within the file Fc The curve to which the report point applies identified as the flow cell Biacore T100 Software Handbook 119 version AB Data presentation tools 120 Time Report point time in seconds from the start of the cycle Window Report point time window in seconds AbsResp Absolute response in RU calculated as the mean value over the time window SD Standard deviation of data points in the time window calculated as 1 2 SD y fc o where n number of points and y response in RU Slope Slope during time window in RU s t calculated as EV e 0 7 x 3 slope 3 Nx x LRSD Alignment of slope to a straight line regression coefficient calculated as LRSD 2 n 2 where 2 a Di Gee Qo Yi y 2 i x Baseline Yes for report points defined as baseline Otherwise No RelResp Relative response
51. 1 1 Selecting sensorgrams for display The selector bar at the top of the window controls which sensorgrams will be displayed E Curve Name Fe 4 v 4 Assay Step Purpose lt Overlay gt Y v Eq Cycle Overlay Y v e Curve name lists the type of sensorgram active reference reference subtracted and solvent corrected where applicable e Assay Step Purpose filters the sensorgram according to the assay step purpose e Cycle lists all the cycles in the result set When multiple files are open cycles are identified with two numbers one for the file in the result set and one for the cycle within the file thus cycle 1 10 is the 10 cycle in the first file added to the result set cycle 2 4 is the 4 cycle in the second file and so on For each option click the browse buttons to browse backwards or forwards through the list one item at a time Click the selector button to open the list for selecting one or more items Drag with the mouse or use shift click to select contiguous multiple items Use control click to select non contiguous multiple items To accept a selection click anywhere outside the list or press Enter 7 1 2 Removing data To remove data from the display mark the section to be removed by dragging with the right mouse button then choose Cut from the right click menu The data will be removed from the current sensorgram window only no other windows or evaluation items will be affected Biacore T100 Software
52. 1052 Yes 0 0 baseline 2 54 0 5 38982 8 0 338 0 1805 0 01771 No 28 6 stability 3 140 5 380041 0 3052 0 1576 0 08816 Yes 0 0 baseline 3 54 0 5 390260 0 1405 0 0721 0 04392 No 21 9 stability 4 14 0 5 3390321 0 1959 0 09877 0 07261 Yes 0 0 baseline 4 54 0 5 380435 0 1014 0 05346 0 01885 No 17 5 stability 5 140 5 39045 7 0 11 0 04598 0 07666 Yes 0 0 baseline 5 54 0 5 390608 0 0181 0 003348 0 01899 No 15 1 stability 6 14 0 5 390306 0 1029 0 05246 0 03448 Yes 0 0 baseline 6 54 0 5 39052 1 0 02661 0 009375 0 02237 No 21 5 stability ti 14 0 5 330305 01271 0 06138 0 06077 Yes 0 0 baseline 7 54 0 5 390521 0 0362 0 01183 0 03203 No 21 7 stability 8 140 5 390305 0 1047 0 04788 0 06065 Yes 0 0 baseline 8 540 5 33052 0 0 03244 0 01417 0 0208 No 21 5 stability g 14 0 5 380326 0 1106 0 05 0 06601 Yes 0 0 baseline 9 54 0 5 39053 7 0 02146 0 007478 0 01819 No 21 1 stability 10 14 0 5 39030 7 0 08505 0 03281 0 06581 Yes 0 0 baseline 10 54 0 5 39051 3 0 04838 0 009263 0 0505 No 20 6 stability 11 14 0 5 39028 7 0 09605 0 0452 0 05092 Yes 0 0 baseline 11 54 0 5 33048 4 0 02017 0 001333 0 02238 No 20 7 stability 12 14 0 5 380123 0 08951 0 0442 0 03833 Yes 0 0 baseline 12 54 0 5 39033 6 0 02868 0 00904 0 02589 No 21 3 stability 13 14 0 5 380058 0 1055 0 04397 0 07375 Yes 0 0 baseline 13 54 0 5 39027 0 0 02513 0 00904 0 02078 No 21 2 stability 14 14 0 5 39002 0 0 09961 0 05156 0 02777 Yes 0 0 baseline 14 54 0 5 390231 0 04357 0 020
53. 2 3 Ligands for capture samples and enhancement reagents should be prepared in each of the running buffers tested Separate rack positions will be created for samples in each buffer for example running buffer scouting with 4 buffers and 5 samples will require 20 sample positions 4 Sample parameters Enter the samples to be tested in the buffer scouting The scouting procedure will work through the sample table for the first buffer before switching to the next buffer 5 System preparations Check the System preparations options as required see Section 4 2 4 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Buffer scouting results When the wizard run is completed the results are opened automatically in the Evaluation Software In addition to the general predefined plots Section 6 4 plots of binding and stability against sample are created to visualize the behavior in the different buffers Biacore T100 Software Handbook Version AB Application wizards la Stability vs Sample 5 x z Curve Name Fe 2 assay Step Purpose Sample In 4 Sample Overlay v Tools gt Stability vs Sample T Zoom Lock value
54. 200 400 600 800 1000 1200 1400 1600 1800 5a Ye Report point table 4780 003 003 00 T9702 No Keyword table Status bar JMeesemm o mes Semple comparten temperature curent 25 C sel 25 C Running standby remaining ine 34 days p e The menu and toolbar provide access to control commands e The sensorgram window displays the sensorgrams for the current run or the currently open file The report point table lists report points for the currently displayed cycle Report points are defined automatically custom report points can also be added in methods e The keyword table lists keywords for the currently displayed cycle Keywords are defined automatically in wizard runs or in the method for method runs e The status bar displays the instrument status including the temperature of the detector and the sample compartment and the type of chip docked e The event log records settings at the start of the run and instrument control events during the run The event log is displayed in a separate window opened by clicking on the Event Log button at the right of the toolbar 6 Biacore T100 Software Handbook Version AB Control Software general features 2 2 1 On line help On line help is available at any time from the Help menu Context specific help for dialog boxes is provided through Help buttons in the boxes 2 3 Basic operation File Open New Wizard Template
55. 37 2 5000 401 2552 2 0257 38 9 8000 394 5078 1 8721 39 39 0000 366 3971 1 3554 40 156 0000 281 2220 0 5169 41 625 0000 171 8457 0 1299 42 2500 0000 99 9902 0 0380 154 43 10000 000 58 7090 0 0133 Calculated Concentration Beq o in Parameters KD 63 733 nM Standard error KD 8 65 nM 0 5 t t t t 1 Ch 0 007 nM 1 10 100 1000 10000 100000 Concentration A nM Help lt Back Finish Cancel The table shows the numerical results for the sample series Samples that lie outside the range of the calibration curve are marked as N A not applicable in the column for Calc Conc Beq The plot panel shows the sample measurements with a line fitted according to the equation for 1 1 affinity see Section 11 1 2 Right click on a point to exclude it from the fitting Notes The plot of free B against total A is presented by default with a logarithmic scale on the x axis In this form the equilibrium constant KD is given by the concentration of A at the inflection point in the fitted curve Zero values cannot be plotted on a logarithmic scale If you have included a sample with zero concentration of A in the sample series and want to display this point on the plot choose Scale from the right click menu in the plot panel and set a linear scale for the x axis The intercept of the fitted curve on the y axis represents a fitted value for the parameter ConcB This value should be the
56. 5 188 i je 21 501 2437 01 1 813 3 18 54 1 3 Avg 18 67 0 9643 bd ii i i 600 4 22 501 2438 00 1 737 5 23 01 1 23 501 2438 01 1 729 3 23 55 1 Avg 23 28 1 637 400 4 26 501 2439 01 1 812 8 18 57 1 27 501 2439 01 1 822 6 18 05 1 x T CTI 200 D E A SA A R RA R a Mi i i 0 10 20 30 40 50 60 70 80 90 100 zl Measured concentration ng ml Sample concentrations are calculated from the nearest preceding calibration curve or the nearest following if there is no preceding curve The left hand panel lists the results sorted by sample ID with averages and CV values for each sample ID The column Calc Conc gives the concentration calculated for the original sample obtained as the measured concentration multiplied by the dilution factor Concentrations for samples that give a response outside the range of the calibration curve are listed as N A not applicable Select a row in the table to highlight the corresponding point on the plot The right hand panel shows the calibration curve and samples calculated from it for the currently selected sample Sample points represent the measured concentrations not the values calculated for the original samples Right click on a sample row in the table or a sample point on the curve and choose Exclude Cycle to exclude that sample from the average sample calculation Excluded cycles are shown as open symbols and are marked in the table with red strikethrough text Bi
57. Afassay Step Purpose lt Overly gt H lt Cycle number oves n Tools v RU Baseline Sample Pza Evalue 1 Y Value p D Zoo Lock 1 378704 2 947 337885 1 Evaluation explorer M 1180 37884 4 Curve Name Fo 4 3 n edo Step Purpose lt Overay m m Cycle number ove m m Binding level T Zoom Lock Rake araue 0 3 Er 07 Er s iy E 33 57 87 Work area Status bar 3 Biacore T100 Software Handbook 95 version AB Evaluation software general features e The menu and toolbar provide access to the evaluation functions e The Evaluation explorer at the left of the screen lists the evaluation items plots sensorgrams and other result displays that have been created in the current session Double click on a folder in the evaluation explorer to expand or collapse the folder Click on an item to display it in the work area Right click in the explorer area for options for adding new items right click on an item for additional options relating to that item e The work area displays the currently open items Each item is shown in a separate window that can be moved resized or closed independently of the other items e The status bar indicates the progress of current operations 6 2 Opening files To open a res
58. Affinity for steady state affinity evaluation or Kinetics for kinetics evaluation when you are satisfied with the curves 3 Affinity only If you choose to evaluate steady state affinity the next dialog gives a preview of the plot of steady state response against concentration with the option to adjust the selection of data used to calculate response values EB kinetics Affinity Select Affinity Data Create xj Note The data set illustrated here is not in fact suitable for affinity analysis Sensorgrams should approach steady state at all concentrations for reliable determination of affinity constants The top panel shows the plot of Reg against C based on average response values over the region marked on the sensorgrams Click Settings to adjust the region used for calculation of Reg values Biacore T100 Software Handbook 133 version AB Kinetics and affinity analysis 134 E Report Point Settings xj Calculate response at position fi 0 seconds before injection stop with window v Help Cancel Click Next gt when you are satisfied with the data selection seconds In the next dialog applicable to both kinetics and affinity you select the fitting model and perform the fit The same data can be fitted repeatedly to different models or to the same model with different settings ES Kinetics Affinity Fit Kinetics Create xi Modet 1
59. Biacore T100 hana Software Handbook BIACORE Ciba TT ere es Biacore T100 Software Handbook Edition February 2006 Biacore T100 Software Handbook Edition February 2006 Version AB Copyright O Biacore 2006 All rights reserved No parts of this publication may be reproduced without written permission from Biacore Biacore AB reserves the right to make changes to the design and specification of Biacore T100 and to the content of this Handbook without prior notice Minor differences may occur between illustrations in this Handbook and the appearance of the product Biacore is a registered trademark of Biacore AB Microsoft and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Biacore AB Rapsgatan 7 SE 754 50 Uppsala Sweden www biacore com Contents 1 Introduction 1 1 1 2 1 3 System overview Support for use in regulated environments Associated documentation 2 Control Software general features 2 1 2 2 2 2 1 2 3 2 3 1 2 3 2 2 3 3 2 3 4 2 3 5 2 3 6 2 3 7 2 4 2 4 1 2 4 2 Operational modes User interface On line belp Basic operation Selecting cycles and sensorgrams File menu Edit menu View menu Run menu Tools menu Right click menus File storage Wizard templates and methods Result files 3 Manual run 3 1 3 1 1 3 2 3 3 3 4 Preparing for a manual run Instrument pre
60. Concentration Sample id MW Da Back Next gt Close Enter the details of the samples for the mass transfer control experiment Each sample will be analyzed at fixed flow rates of 5 15 and 75 pl min with a contact time of 1 minute and dissociation time of 2 minutes Use an analyte concentration that gives readily measurable initial binding rates Mass transfer limitation is not affected by analyte concentration but dependence of binding rate on flow rate may be difficult to detect if the binding rate is too low or too high Biacore T100 Software Handbook Version AB Application wizards 4 6 2 Linked reactions control Tro Kinetics Linked Reactions Samples xj Samples Concentration Concentration Sample id MW Da lt Back Next gt Close Enter the details of the samples for the linked reactions control experiment Each sample will be analyzed at a flow rate of 10 pl min with fixed contact times of 0 5 3 and 10 minutes and a dissociation time of 10 minutes Use one or more fairly high analyte concentrations preferably so that steady state is approached or reached within the shortest contact time The experiment is easiest to interpret if the interaction is maintained at steady state for varying lengths of time so that the starting response for dissociation is constant 4 6 3 Evaluation of control experiments When the wizard run is completed the results are
61. Cone If weight based units are used a molecular weight for the analyte must be specified in the variable MW As a recommendation there should be a concentration series with at least five non zero analyte concentrations and one zero concentration At least one of the non zero concentrations should be measured in duplicate Although kinetic and affinity evaluation can be applied to runs with fewer sensorgrams the results will generally be less reliable if these recommendations are not followed 5 10 3 Thermodynamics See Chapter 10 for a description of thermodynamics evaluation Thermodynamics evaluation requires that kinetics or affinity see Section 5 10 2 is determined at two or more temperatures 5 10 4 Affinity in solution See Chapter 11 for a description of affinity in solution evaluation At least one assay step is required with purpose Calibration and one with purpose Sample Both assay steps must be connected to a cycle type that includes one Sample command The two assay steps will normally be connected to the same cycle type Samples in the Calibration step must have concentrations specified in the variable ConcB calibration At least two different concentrations are required for linear calibration curves and at least four for 4 parameter fitting These samples should contain only component B Samples in the Sample step must have concentrations specified in the variables ConcB fix and ConcA variable At least 3 samples w
62. Displaying tbe report point table 8 Concentration analysis 8 1 8 2 8 2 1 8 2 2 8 2 3 8 2 4 Biacore T100 Software Handbook version AB Requirements for concentration evaluation Evaluating concentration analysis Calibration curves Control samples Samples Evaluating combined result sets Contents 87 87 88 89 90 90 91 95 95 96 96 96 97 98 98 99 100 101 103 103 104 105 105 109 109 110 110 111 113 114 115 116 117 118 119 119 123 123 123 124 125 127 128 iii Contents 9 Kinetics and affinity analysis 129 9 1 Kinetics and affinity determination 129 9 1 1 Requirements for kinetics and affinity evaluation 129 9 1 2 Evaluating kinetics and affinity 130 9 1 3 Multiple ligand densities 137 9 2 Curve fitting principles 138 9 2 1 Fitting procedure 139 9 2 2 Local and global parameters 140 9 2 3 Parameter significance 141 9 3 Predefined models 144 9 3 1 Kinetics 1 1 binding 145 9 3 2 Kinetics Bivalent Analyte 146 9 3 3 Kinetics Heterogeneous Analyte 147 9 3 4 Kinetics Heterogeneous Ligand 149 9 3 5 Kinetics Two State Reaction 151 9 3 6 Affinity Steady State 1 1 152 9 4 Creating and editing models 153 9 4 1 Interaction models for kinetics 154 9 4 20 Equation models for kinetics 158 9 4 3 Models for steady state affinity 160 10 Thermodynamic analysis 161 10 1 Background 161 10 1 1 Equilibrium thermodynamics 161 10 1 2 Transition state th
63. a Y Value 60 4 B2u 16n 33 7 B2u 16n 34 6 B2u 32n 54 6 55 B2u 32n 54 2 M B2u 8n 30 3 B2u 8n 31 4 50 2 a E Bast 8 HBS EP 1X P20 1 e i i HBS EP10x P20 40 5 o E 3 2 354 304 25 T t t t t t 1 Sample 4 5 Assay wizards 4 5 1 Binding analysis The binding analysis wizard supports injection of up to four samples in series in addition to ligand capture enhancement and regeneration steps This wizard is suitable for analysis of applications like multi component complex formation and pair wise epitope mapping as well as simple applications like screening for binding partners to an immobilized ligand 1 Injection sequence Choose the injection sequence for the binding analysis see Section 4 2 1 Up to four sequential sample injections may be included in each cycle 2 Setup Specify the flow path and start up cycles see Section 4 2 2 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 One sample injection panel will be created for each sample injection in the injection sequence Biacore T100 Software Handbook 47 version AB Application wizards 48 4 Sample parameters T Binding Analysis Samples The sample table contains one column for each sample injection in the injection sequence New rows are created as you enter data in the table The illustration above shows how the wizard could be used to s
64. a presentation tools 116 7 2 2 Table functions The table to the right of the plot area lists values for the points in the plot Excluded points are shown struck out in red text You can exclude or include cycles from the right click menu in the table area in the same way as from the right click menu in the plot The table also allows you to exclude or include multiple cycles in a single operation By default the table shows x and y values and is sorted in ascending order of x values Click on the header row to select the sort value and to change the sort order Sorting the table does not have any effect on the plot display Choose Tools Table columns to select columns that will be displayed in the table You can also change the order in which columns will be displayed using the Move up and Move down buttons the top of the column list represents the left hand column in the table Table Columns X Move Up YA alue Ranking Move Down CO Cycle number O CycleType AssayStep CO AssayStepPurpose CJ Temp L1 Sample OFe Biacore T100 Software Handbook Version AB Data presentation tools 7 2 3 Fitting curves to points Choose Curve Fitting under the Tools button to fit lines to the points in the plot You can choose linear or curved 4 parameter fitting functions The numerical fitting results are displayed in an extra panel below the plot If Fit by color is checked each color will be fitted to an indepen
65. acore T100 Software Handbook 127 version AB Concentration analysis 128 8 2 4 Evaluating combined result sets If you use the Append File function to combine result sets from separate runs concentration analysis can be evaluated provided that the conditions specified in Section 5 10 are fulfilled in the combined set The software does not check the validity of any evaluation applied to a combined result set so that it is your responsibility to determine that the evaluation results are meaningful It is for example in principle possible to append a kinetic analysis result file to a concentration analysis and then apply concentration analysis evaluation provided that the report point used for the calibration curve exists in the cycles from the kinetic run calculated concentrations will be reported for these samples In order to ensure that concentration analysis is correctly evaluated in combined result sets make sure that all files that contribute to the combined result set are derived from concentration analysis runs Provided that each file starts with a calibration curve that is not excluded from the evaluation the results will be calculated within the respective files even in the combined result set However if calibration curves at the beginning of files are missing or excluded there will be overlap between the individual file sets and some samples from one file will be evaluated on the basis of calibration curves from another fil
66. al fitting function for continuous curves and is recommended for most purposes Use a linear function only if you have good reason to expect the calibration curve to be a straight line See Section 7 2 3 for the equations for fitting functions The left hand panel lists the details of the calibration curve data with concentration and response or slope values for all calibration points The Calc conc column lists the concentration corresponding to the actual response value as determined from the fitted calibration curve Select a row in the table to highlight the corresponding point on the calibration curve The calibration curves are shown in the right hand panel Choose the curve to display from the list at the top of the panel Each calibration curve is constructed from the cycles in an assay step with the purpose Calibration Right click on a curve or calibration point for options for excluding calibration curves or single cycles from the evaluation Excluded cycles are shown as open symbols and excluded curves are shown as broken lines The excluded points are marked in the left hand panel with red strikethrough text You can also choose to display sensorgrams for individual calibration cycles from the right click menu Note The sample name is ignored when the calibration curve is constructed For wizard based runs the sample name will be the same for all calibration cycles but different names can be introduced either in method based run
67. ally be unchanged throughout the run Biacore T100 Software Handbook Version AB Application wizards 4 4 3 Buffer scouting The buffer scouting wizard helps you to test the effect of up to four different buffers on your assay using the buffer selector valve to switch running buffers 1 Injection sequence Choose the injection sequence for the buffer scouting see Section 4 2 1 2 Assay setup x Detection Use the same flow path for all buffers l 7 Vary flow path with buffer Startup IV Run startup cycles Solution p ummy Number of cycles 3 E Buffers Buffer Name Specify the flow path and start up cycles The flow path can either be chosen explicitly in which case the same flow path will be used for each buffer or set to vary with the buffer in which case a single flow cell will be used for each buffer with flow cells 1 2 3 and 4 for buffers A B C and D respectively If start up cycles are chosen separate rack positions will be created for the start up sample solution in each buffer tested and the start up cycles will be run at the start of each buffer test Specify the buffers you want to test You can enter up to four different buffers The buffers will be tested in the order given Biacore T100 Software Handbook 45 version AB Application wizards 46 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4
68. aluation of concentration kinetics or affinity and may therefore also be used to hide injections from the predefined evaluation facilities The injected solution contact time and flow rate can be set as variables Evaluation variables can also be defined for General commands see Section 5 6 2 Inject and recover command This command recovers analyte that is bound to the sensor surface and is intended for use in applications where the bound material is analyzed further Several features of the command are designed specifically for integration of Biacore analysis with mass spectrometry MS Most of the parameters for the command can be set as variables This command can only be used for injection over all four flow cells normally the same ligand should be immobilized in each cell Commands Report Points Capture Settings for InjectAndRecover 1 Sample solution Sample Method Variables Insert Command Set property as variable Contact time 180 sg C Sample sok Remove From Cycle ample solution Flow rate 5 pimin O Contact time s Move Up C Flow rate l min Flow path 1234 r wash solution Move Down O Recovery solution Wath solution 0 5 TFA L1 Deposition solution IniectAndRecover 1 eren SER EI Deposition solution volume Incubation time o fs Deposition solution 50mM NH4HCO3 Deposition solution volume fo ul Number of repetitions 5 gt The command initiates a sequence of
69. alyte binding capacity of the first Rmax1 ligand RU Rmax2 Analyte binding capacity of the Rmax2 second ligand RU Conc Analyte concentration M Conc Tc Flow rate independent component of tc the mass transfer constant Flow Flow rate pl min f Kt Mass transfer constant tc f1 3 RI Bulk refractive index contribution in RI the sample 150 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis 9 3 5 Kinetics Two State Reaction This model describes a 1 1 binding of analyte to immobilized ligand followed by a conformational change that stabilizes the complex To keep the model simple it is assumed that the conformationally changed complex can only dissociate through the reverse of the conformational change A B AB AB Note that conformational changes in ligand or complex do not normally give a response in Biacore A good fit of experimental data to the two state model should be taken as an indication that conformational properties should be investigated using other techniques e g spectroscopy or NMR rather than direct evidence that a conformational change is taking place Model parameters are Obtained from kal Association rate constant for analyte Fitted binding M s kd1 Dissociation rate constant for analyte Fitted from the complex s t ka2 Forward rate constant for the Fitted conformational change s kd2 Reverse
70. ample injection and recovery operation Te Method Builder Main Oven i crests asa tp by Genetal Setings ug te butana Seo Surface conditioning Conditioning Conditioning 3 times as entered 2 Select an assay step by gt clicking on it Edit properties Sample Inject and Recover Sample Inject and Recover _1 time as entered Recurrence Surface conditioning Repeat assay step within zj Conditioning s Cite F uis Distribute F zj occurrences evenly Conditioning s T Aun assay step once first I Run assay step once last Numberofteplicales Assay step preparations BOX times Temperature ps As entered 123123 Butter fs C order 112 233 C Random Biacore T100 Software Handbook 91 version AB Methods The cycle for recovery of bound analyte contains just one single InjectAndRecover command Additional commands are usually not needed since the recovery component of the InjectAndRecover command serves as a regeneration step see Section 5 6 1 Te Method Builder Main The Inject and Recover cycle type regulates sample injection washing of the fluidic system and recovery of the material that is captured on the pene surface shi one cycle the sample injection end recover sequence can be D1 Deposition solution D1 Deposition solution volume All parameters in the InjectAndRecover command are fixed check the appropriat
71. ards 36 Choose the way in which immobilization will be controlled e If you choose Aim for immobilized level you specify a target level The immobilization procedure will attempt to reach this level as described below e If you choose Specify contact time and flow rate enter the settings in the respective fields e If you choose Blank immobilization the surface will be activated and deactivated in accordance with the immobilization method but no ligand will be injected Enter the ligand name To dilute the ligand solution immediately before injection check Dilute ligand and enter a percentage value and a solution name This option can be used for ligands that have limited stability in immobilization buffer and that are diluted from a stock solution just before immobilization A setting of 90 will mix one part of ligand solution with 9 parts of the specified diluent Aiming for immobilized level The option Aim for immobilized level injects a pulse of ligand over the unactivated surface in order to estimate the rate of preconcentration The surface is washed to remove traces of ligand and then activated The procedure then uses ligand contact times based on the preconcentration estimate to attempt to reach the specified target level If preconcentration is either too fast or too slow to permit the target level to be reached this will be reported and immobilization will not be performed The preconcentration injection injects 10
72. art of a run when the wizard template is used Regeneration scouting results Regneration scouting results are presented in the Control Software when the run is completed Note This result presentation is not shown if the run is opened in the Evaluation Software T Regeneration Scouting Results xd Trend Chart Sensorgrams 4 Sample Response Baseline RU RU 740 4 r 47450 730 4 Min RN 720 4 ere ep 47400 710 ds 47350 874 s 1 AAAA 2 Fi f 890 4 47300 g 9 6804 P d 8 H 47250 670 660 1 47200 a Ame a a iS 640 4 t t t r t t t r t t 47150 0 3 6 3 12 15 18 21 24 Cycle Display Sensorgram L lstcycle Conditions feran Fe 4 The Trend chart tab shows the results as a plot of baseline and sample response for each cycle in the run grouped by regeneration conditions Conditions are identified in tool tips for the data points place the cursor on a point for a couple of seconds to display the tool tip Biacore T100 Software Handbook Version AB Application wizards Note Report points are set before the sample injection for baseline and shortly after the sample injection for sample response Thus the points for the first cycle indicate the starting values while those for subsequent cycles each indicate the effect of the previous cycle Check 1st cycle to include the starting values derived from the first sample cycle in the
73. art up cycles are treated separately from analysis cycles in the evaluation software 4 2 3 Injection parameters The Injection parameters dialog specifies details of the injections selected in the Injection sequence Ts Binding Analysis Injection Parameters xi Capture Solution ligand Contact time eo s Flowrate 10 ul min Stabilization period fo s Sample Contact time feo s Flow rate o pl min Dissociation time eo s M Enhancement Solution secondary Ab Contact time eo s Flowrate 10 pl min m Regeneration Solution ea solutiorl High viscosity solution Contact time 36 s Flowrate 30 ul min Stabilization period fo s Help lt Back Next gt Close Details of this dialog box may vary according to the injections selected and the particular wizard Some features may be generalized Biacore T100 Software Handbook 25 version AB Application wizards 26 Parameter limits Flow rates can be set between 1 and 100 pl min in increments of 1 pl min Permitted ranges for injection contact times are determined by the flow rate together with the limits for injected volumes which are 2 350 pl for normal solutions and 5 100 pl for viscous regeneration solutions see below Note The injected volume of solution is determined by the combination of flow rate and contact time rounded up to the nearest whole number At low flow rates th
74. ath and start up cycles see Section 4 2 2 Only reference subtracted detection using either Fc2 1 or Fc4 3 is available for kinetic analysis Start up cycles are recommended for kinetics experiments to ensure that the initial response drift that may occur with a new chip does not interfere with the first measurements Biacore T100 Software Handbook Version AB Application wizards 3 Injection parameters T Kinetics Affinity Injection Parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 The sample injection for kinetics measurement has an additional setting for dissociation time This is the time for which dissociation will be monitored after the end of the injection without disturbances from flow system washing procedures 4 Sample parameters To Kinetics Affinity Samples Enter the details of the samples for kinetic or affinity determination For each analyte a zero concentration sample and at least five non zero concentrations one of which is run in duplicate are Biacore T100 Software Handbook 55 version AB Application wizards 56 recommended Concentrations are entered in the left hand Concentration column either in molar or weight based units Choose the concentration units from the pull down list in the column header If a weight based concentration unit is chosen e g ng ml a molecular weight must also be specified When a molecular weight i
75. ation curve will be run at the beginning of the assay and at the specified interval Thus specifying a repeat every 15 sample cycles and running 35 samples will result in calibration curves at the beginning and after samples 15 and 30 If the Repeat calibration box is not checked calibration will be run once at the beginning of the assay Enter the concentrations for the calibration points on the curve You must enter at least two concentrations for the calibration curve Two concentrations are sufficient for a linear calibration curve but if you intend to use the recommended four parameter fitting function for the calibration curve you need at least four points To run replicate concentrations enter the same concentration on multiple rows Calibration points will be run in the order entered You can choose a different concentration unit if required from the pull down list in the table header Biacore T100 Software Handbook 51 version AB Application wizards 52 5 Control samples Te Concentration Analysis Control Samples xj M Control sample definition IV Run control samples v Repeat control sample s every fis sample cycles M Control samples Expected conc Control Sample id ug ml 1 High 45 2 Low Help lt Back Next gt Close Specify the details of control samples and the frequency with which they should be run You may choose not to run control samples at all
76. ayed at the start of a run when the wizard template is used Evaluation Section 10 2 describes how to evaluate thermodynamic assays 4 6 Control experiment wizards Two control experiments are currently supported by wizards both for kinetic analyses e The Mass transfer control experiment analyses the interaction of one or more analyte concentrations at three different flow rates to establish whether the observed binding rate varies with flow rate A dependence of binding rate on flow rate indicates that the binding is limited to some extent by mass transfer of analyte to the sensor surface Biacore T100 Software Handbook 59 version AB Application wizards 60 e The Linked reactions control experiment analyses the interaction of one or more analyte concentrations for different contact times to identify a particular kind of deviation from a 1 1 binding mechanism Variation of the dissociation behavior after the end of the injection with the contact time indicates that the observed binding consists of at least two processes one where the analyte binds to the surface and a second where the surface attached complex undergoes a stabilizing alteration such as a conformational change The control experiment wizards have the same structure as the kinetic analysis wizard but differ in the details of the sample parameters step 4 6 1 Mass transfer control T Kinetics Mass Transfer Samples x Samples Concentration
77. c Flow rate independent component of the mass transfer constant Fitted f Flow rate pl min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in Fitted the sample The following values are presented in the report Calculated as ka Association rate constant M 1s ka kd Dissociation rate constant s kd KD Equilibrium dissociation constant M kd ka Rmax Analyte binding capacity of the Rmax surface RU Conc Analyte concentration M Conc tc Flow rate independent component of tc the mass transfer constant Flow Flow rate pl min f kt Mass transfer constant tc 13 RI Bulk refractive index contribution in RI the sample Biacore T100 Software Handbook version AB 145 Kinetics and affinity analysis 146 9 3 2 Kinetics Bivalent Analyte This model describes the binding of a bivalent analyte to immobilized ligand where one analyte molecule can bind to one or two ligand molecules The two analyte sites are assumed to be equivalent The model may be relevant to studies among others with signaling molecules binding to immobilized cell surface receptors where dimerization of the receptor is common and to studies using intact antibodies binding to immobilized antigen As a result of binding of one analyte molecule to two ligand sit
78. c analysis Before thermodynamic analysis can be performed for a set of data the kinetics and or affinity must be evaluated at each temperature Create a separate Kinetics Affinity evaluation item at each temperature used in the run using the same fitting model for each item see Section 9 1 2 When all required kinetic and affinity evaluation items have been created click Thermodynamics on the toolbar EG Thermodynamics Select Kinetic Data Create xj m Import Kinetic and Affinity Evaluations Sample b2micro X I ne jy state affinity Evaluation Temperature C al Model Description ka 1 Ms kd 17s KD M b2micro 4 4 1 1 Binding 2 296E 5 3839E4 1 572E 8 b2micro 12 12 1 1 Binding AUU3E 5 6 615E 4 1 553E 9 b2micro 19 19 1 1 Binding 5 070E 5 1410E3 2781E 9 b2micro 26 26 1 1 Binding amp 528E5 2874E 3 4 402E 8 b2micro 33 33 1 1 Binding 3 636E 5 6 664E 3 6 915E 9 b2micro 40 40 1 1 Binding 1 657E 6 1 852E 2 1 124E 8 Check All Uncheck All Help Back Next Cancel Choose the sample and the fitting model in the pull down lists You may only choose one sample and you should only choose one fitting model Options for the model are 1 1 kinetics steady state affinity recommended or All If you choose All it is possible to combine data from different fitting models in the same evaluation however values for thermodynamic constants are in al
79. can continue to the next step Evaluation variables may be left blank if desired 5 9 3 Cycle run list When you have completed the variables table click Next to view a complete summary of the cycles that will be performed in the run This view is for information only and cannot be edited Check through the cycle list to confirm that the variable tables are correctly filled in Biacore T100 Software Handbook 83 version AB Methods 84 T BufferChange Cycle run list Gycle Assay step ame Sample 1 Solution Sample 1 BatchNo Sample 1 Conc uv sample 1 Dilution 44387 GRH 65103 Click Overview to display the method overview Section 5 3 5 9 4 System preparations Choose which preparation steps should be executed before the method starts System Preparations M L Temperature settings Analysis temperature PL sample compartment temperature 15 System preparations are equivalent to those for wizard based runs see Section 4 2 4 The temperature settings are taken from the first assay step in the method and cannot be changed here they are shown for information only Biacore T100 Software Handbook Version AB Methods 5 9 5 Rack positions See Section 4 2 5 for a description of the Rack positions dialog box 5 9 6 Prepare Run Protocol See Section 4 2 6 for a description of the Prepare Run Protocol dialog box 5 9 7 Starting the run When the positions are finalized th
80. ctively All settings except temperature and choice of microplate and or reagent rack can be changed during the run Commands are placed in a queue if the instrument is busy when a command is issued queued commands that have not yet been started can be edited or deleted from the queue The results of a manual run are saved in a normal result file and can be evaluated in the Evaluation Software There are however no predefined keywords associated with the run and the results cannot be evaluated with the tools for concentration kinetics affinity thermodynamics or affinity in solution 3 1 Preparing for a manual run 3 1 1 Instrument preparations The integrated instrument preparation steps that are included with wizard and method based runs are not supported for manual run The instrument should therefore be prepared using options from the Tools menu 1 Dock the chip that you want to use and immobilize ligand on the surface see Section 4 3 2 if this has not already been done 2 Choose Tools Prime to flush the flow system with fresh buffer 3 Choose Normalize from the Maintenance Tools section of Tools More Tools if the detector has not been normalized since the chip was docked In many cases the detector will have been normalized in connection with ligand immobilization However you may need to perform the operation again if the chip has been undocked and re docked after immobilization 4 Choose Tools Set Temperature and se
81. curring step is set in the properties of that step and is not affected by the number of replicates at the higher level 5 5 3 Recurrence An assay step can be set to recur at a specified interval within the context of another step The recurrence can be specified as Every n cycles so that the number of occurrences will depend on the number of cycles in the assay step or Distribute n occurrences evenly in which case the number of occurrences is fixed and they are distributed as evenly as possible among the cycles in the assay step In addition the recurring step can be specified to be executed at the beginning and or end of the step in which it is set to recur If the higher level step is run in replicate a recurring step is distributed among the total number of cycles including replicates This is illustrated by the calculations below for a recurring step set to Every 5 cycles Top level step Number of cycles 10 20 10 Number of replicates 1 1 2 Total number of cycles 10 20 20 Number of recurrences for the nested step 2 4 4 Biacore T100 Software Handbook Version AB 5 6 Cycle types Biacore T100 Software Handbook version AB Methods 5 5 4 Assay step preparations Temperature This value determines the analysis temperature for the assay step The setting will also control the sample compartment temperature if the appropriate option is checked under General Settings Section 5 4 If the ac
82. d in the dialog box that appears when you click Inject Mix amp Inject mixes two specified solutions and performs an injection of the mixture Details are entered in the dialog box that appears when you click Mix amp Inject Wash washes the flow system but not the sensor surface The wash solution is specified in the dialog box that appears when you click Wash Select an injection and use the Edit Delete Move up and Move down buttons to edit the injection details remove the injection from the method and change the order of injections in the method Custom methods are stored in the immobilization wizard template if you need the same or slightly modified method in a different template save a copy of the template and then edit the method 2 System preparations Check the System preparations options as required see Section 4 2 4 3 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 4 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Immobilization results The results of an immobilization run are summarized in the Control Software and logged in the Chip Properties see Section 2 3 4 when the run is completed Note This result presentation is not shown if the run is opened in the Evaluation Software
83. d in startup and sample steps Contains sample and regeneration 88 Biacore T100 Software Handbook Version AB Methods 5 11 3 Low molecular weight compounds Methods are provided for both kinetics and screening of low molecular weight compounds The essential addition to wizard based counterparts is the inclusion of a Solvent Correction assay step repeated at interval throughout the run Concentration unit nM Startup Data collection rate 10Hz Startup LMW kinetics 3timesasentered Sample compartment temperature 25 C Detection Dual Sample Sample Lmw Temperature 25 C Solvent correction inr t even correction solvent correction 1 time as entered Before every 20 cycles Control sample E control sence mw kretics 1 time as entered Before after every 10 cycles fH Sample 1 varies by cycle 60s 600s E Cary over control 1 E Report points This assay step is connected to a cycle type that includes 8 Solvent Correction commands for injection of 8 different solvent concentrations see Section 6 7 This cycle is used in solvent correction steps Contains injection of eight different solutions Solvent correction 8 Biacore T100 Software Handbook 89 version AB Methods 90 5 11 4 Screening Screening applications suitably include a carry over control injection to identify potential carry over problems from sticky comp
84. d in the report Kinetics and affinity analysis Calculated as ka1 Association rate constant for the first kal site M 1s 1 kd1 Dissociation rate constant for the first kd1 site s t ka2 Association rate constant for the ka2 second site RU s 1 kd2 Dissociation rate constant for the kd2 second site s Rmax Analyte binding capacity of the Rmax surface RU Conc Analyte concentration M Conc tc Flow rate independent component of tc the mass transfer constant Flow Flow rate l min f kt Mass transfer constant tc fl 3 RI Bulk refractive index contribution in RI the sample 9 3 3 Kinetics Heterogeneous Analyte This model is intended for analysis of the kinetics of interaction of mixtures of two analytes that compete for the same ligand site Experiments of this kind can be used to deduce kinetic parameters for a low molecular weight analyte that gives a small response from measurements of binding of a competing high molecular weight analyte Response contributions from both analytes are taken into account although the high molecular weight analyte is responsible for the dominant component in the observed sensorgrams Concentrations and molecular weights are required for both analytes If absolute molecular weights are not known relative values can be entered without affecting the outcome of the fitting The model cannot evaluate interactions where the proportions and
85. dent line If this option is not checked all points derived from the same curve will be fitted to a single line igix E Curve Name Fc 4 3 hA v Assay Step Purpose verlay n 4 Cycle number lt Overlay gt T Ii Tools gt Baseline Sample X Value alue P Zoom Lock 18361097 1837 634 1838 078 1849 594 1850 429 1852 558 1854 291 1855 785 1856 687 1857 598 1858 209 1858 666 1859 068 1859 448 1859 688 1860 977 1859 982 1859 932 1859 897 1859 787 1859 712 e th tn t DOO oO 0 0 N buffer 1850 Y Furosemide2 o Furosemide1 1845 T m 0 Absolute response baseline 1 nap 1835 t t t t 1 0 5 10 15 20 25 Cycle number Function Slope Intercept R2 buffer Linear 0 99 1835 29 0 91 Furosemide2 Linear 0 59 1849 45 0 75 Furosemidel Linear 0 48 1850 56 0 77 co D m e NNN Qm For linear fitting the points are fitted to the equation y slope x intercept The equation for a 4 parameter fit is R R y R F d ix A where y and x are the plot coordinates Rp and Rj are fitting parameters that correspond to the maximum and minimum response levels respectively and A4 and A are additional fitting parameters The closeness of fit is reported for linear fitting as the correlation coefficient R2 and for 4 parameter fitting as the chi squared value Choose Cu
86. dit trail is also exported to a separate worksheet if it is present when the GxP module is installed see the separate Biacore T100 GxP Handbook Export to XML To export data to an XML file choose File Export Results to XML This option exports the same data as Results to Excel but creates a text file in XML format file extension xml Details of the XML format may be determined by exporting data from the Control or Evaluation Software and opening the exported file in an XML compatible editor Biacore T100 Software Handbook 171 version AB Affinity in solution Report point table To export the report point table to a tab separated text file choose File Export Report Point Table The exported file has the extension rpt Note If you open an exported report point table in Microsoft Excel make sure that the format for the Fc column is set to Text in the Excel import file wizard The default setting of General for text file import may interpret the flow cell identification for reference subtracted data as a date instead of a text string Rack positions Rack positions can be exported from the Control Software to a tab separated text file in either ASCII or Unicode format using the Menu Export Positions function in the Rack Positions dialog Section 4 2 5 The file contains two lines identifying the microplate and reagent rack settings followed by the contents of the rack positions table with the columns separated by tabs A 2 Im
87. e Check the injections that you want to include The illustration panel shows the sequence of included injections Injections have the following purposes Capture Intended for ligand solution in applications that use a capturing approach to attach the ligand to the surface The same solution will be used for the capture injection in all cycles you cannot vary the captured ligand within the context of one wizard run The flow path for capture solution depends on the settings for detection see Section 4 2 2 Biacore T100 Software Handbook 23 version AB Application wizards 24 Sample This is the sample to be analyzed The solution used for the sample injection is normally different in different cycles and is specified in the sample table at a later stage in the wizard The sample injection is required in all wizards Enhancement Intended for injection of a secondary reagent that binds to analyte on the surface typically used either to amplify the response obtained from the analyte or to enhance the specificity of analyte detection The same solution will be used for the enhancement injection in all cycles Regeneration One or two regeneration injections may be included which may use the same or different solutions The regeneration procedure is the same in all cycles 4 2 2 Assay setup Common features of the assay setup dialog are choice of detection and flow path and specification of start up cycles at the begi
88. e 420 s Flow rate fio yl min C Blank immobilization Flow cell 3 d Immobilize flow cell 3 Method E Amine Aim for immobilized level Ligand ligand 2 IV Dilute ligand C Specify contact time and flowrate Dilute ligand with so EA immob butter C Blank immobilization Target level 10000 RU Wash solution o mM NaOH M Flow cell 4 d HB Immobilize flow cell 4 Method L8 Surface thiol C Aim for immobilized level Ligand fiigand 3 Dilute ligand Specify contact time and flowrate Contact time 420 s Flow rate fio ul min Blank immobilization Help Custom Methods lt Back Next gt Close The choice of Chip type determines the predefined methods that are available for immobilization The type chosen when the chip was docked is chosen by default if you change the chip type you will be able to create and save an immobilization wizard template but you must dock a corresponding chip type before the immobilization can be performed Check the flow cells where you want to perform immobilization For each flow cell set the parameters as follows Choose the immobilization method Predefined methods are provided for standard immobilization chemistries Customized methods can be defined by clicking on the Custom Methods button see below Predefined methods are marked with a T100 icon 8 in the selection lists Biacore T100 Software Handbook 35 version AB Application wiz
89. e In such cases it is important to ensure that the calibration curves and sample analyses in the different files refer to the same analyte and are performed under as far as possible identical condition Biacore T100 Software Handbook Version AB Kinetics and affinity analysis 9 Kinetics and affinity analysis Biacore T100 offers three main functions for analysis of interaction kinetics and affinity e Kinetics and affinity measurements on the sensor surface which determine the interaction characteristics between ligand and analyte Kinetic parameters are evaluated from the association and dissociation phases of the sensorgram and affinity either from the kinetic parameters or from plots of steady state analyte binding levels Req against concentration e Thermodynamic analysis which relies on measurement of either kinetics or affinity at different temperatures e Affinity in solution where the interactants are mixed in known concentrations in solution and allowed to reach equilibrium Biacore T100 is then used to determine the concentration of free interactant in equilibrium with the complex This chapter describes how to evaluate surface bound kinetics and affinity Thermodynamic analysis and measurement of affinity in solution are described in Chapters 10 and 11 respectively 9 1 Kinetics and affinity determination 9 1 1 Requirements for kinetics and affinity evaluation The minimum requirements for evaluation of kinetics or
90. e squared residual the difference between the experimental data and the fitted curve n D za chi squared nap where r is the fitted value at a given point ry is the experimental value at the same point n is the number of data points and p isthe number of fitted parameters For sensorgram data the number of data points is very much larger than the number of fitted parameters in the model so n pen and chi squared reduces to the average squared residual per data point If the model fits the experimental data precisely chi squared represents the mean square of the signal noise In some situations the fitting algorithm may be unable to find a fit for the experimental data with the initial parameter values as specified in the model This may happen typically if the concentration unit is incorrect for example if the unit is set to mM instead of nM in the keyword table On occasion however if can be necessary to adjust the starting values for fitting parameters accessed through the Parameters button in the fitting dialog step 4 in Section 9 1 2 above Biacore T100 Software Handbook 139 version AB Kinetics and affinity analysis 140 9 2 2 Local and global parameters Parameters in the fitting equations are treated as either local or global variables or constants e Local parameters are assigned an independent value for each curve in the data set Typical local parameters are concentration which is differen
91. e as ligand It is assumed that the measurement itself does not significantly disturb the equilibrium in the sample The experimental setup requires a calibration curve with known concentrations of B determined over the same sensor surface in order to calculate the free B concentrations in the samples 11 1 2 Evaluation principles The equilibrium constant for a 1 1 interaction is given by ke B free K p AB Or K A rot E AB B o m AB p AB Biacore T100 Software Handbook 167 version AB Affinity in solution Rearranging gives Kp AB Aq Bi AB A tot Brot AB or AB AB A rot Brot Kp Aa Biot 0 Solving for AB 2 Substituting in the relationship Bfree Btot AB gives 2 Bey _ Brot m Kp e Dus Ky An Bie This equation can be fitted to a plot of Bfree against Atot to calculate a value for Kp 11 2 Requirements for affinity in solution Affinity in solution experiments are run using a method The method must be correctly constructed as described in Section 5 10 4 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command name cannot be edited in the keyword table Refer to Chapter 5 for details of how to construct methods in Method Builder Determination of affinity in solution is not supported by a wizard 11 3 Evaluation of affinity in solution 168 To evaluate affinity in solution measurements open the r
92. e below Note Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray Ifnecessary empty the waste bottle before start of the run 3 2 3 t Running buffer 8j Not in use gj Not in use pj Not in use Menu lt Back Save As Close A Use the controls at the top of the dialog box to set font characteristics for the text The area at the bottom of the dialog box identifies the buffers required in each of the buffer bottles For all wizards except buffer scouting only buffer A is used For the buffer scouting wizard Section 4 4 3 Biacore T100 Software Handbook 31 version AB Application wizards and Method Builder based runs Section 5 4 buffer names are shown in the Prepare Run Protocol dialog The Menu button provides options for saving and printing the wizard template Section 4 2 5 4 3 Surface preparation wizards 32 4 3 1 Immobilization pH scouting The Immobilization pH scouting wizard helps you to find the optimal pH for immobilizing your ligand by testing ligand pre concentration at a range of pH values See the Biacore Sensor Surface Handbook for further details The injection sequence for immobilization pH scouting is fixed 1 Assay setup Tx Immobilization pH Scouting Setup x Detection Fow path Buffers Buffer Name 10 mM Acetate 10 mM Acetate 5 10 mM Acetate 4 5 10 mM Acetate 4 Help
93. e boxes in the Method Variables list if you want to use variable parameters There are no evaluation variables for this command 92 Biacore T100 Software Handbook Version AB Evaluation Software Biacore T100 Software Handbook 93 version AB 94 Biacore T100 Software Handbook Version AB Evaluation software general features 6 Evaluation software general features Biacore T100 Evaluation Software offers general functions for presentation of results as sensorgrams and or report point plots and application specific evaluation functions for concentration kinetics and affinity and thermodynamics measurement There is also a function that corrects for solvent effects that can sometimes distort the results from analyses with low molecular weight analytes that give low response levels and require organic solvents e g dimethyl sulfoxide DMSO to maintain solubility This chapter describes the organization of the evaluation software The various evaluation functions are described in detail in the following chapters 6 1 Evaluation software interface The Biacore T100 Evaluation Software screen is divided into three main regions jobu BOB1 ORC1 7 18_11 050208 kinb2u_V2 blr IES Menu and toolbar Boer iF T i EE MS Cave Name Fo 43 n fessos Step Purpose coetu feces coetus Sensorgram 1170 4 Curve Name Fc WI
94. e list A sample series is defined as all cycles with the same sample name in the assay step s with purpose Sample If you have run multiple calibration curves in the experiment choose the curve to use in the Calibration curve list A calibration curve is defined as measurements from assay step s with purpose Calibration regardless of the sample name If two or more Calibration assay steps are run contiguously with no intervening steps with a different purpose they will be combined into a single calibration curve Note All samples in a series are evaluated against the chosen calibration curve You cannot use different calibration curves for different samples in the same series The table lists the data for the calibration curve The plot panel shows the curve with calibration points as black inverted triangles and sample points as red squares Samples that lie outside the range of the calibration curve are not shown Right click on calibration points to exclude the points from the curve Click Next gt to calculate the results Biacore T100 Software Handbook 169 version AB Affinity in solution 170 A Affinity in Solution Step 2 H557 thrombin 2 x Sample H557 thrombin ConcB 2 nM Constant concentration B 2 nM Sample Information M Concentration Curve Cycle Conc A Response Calc Conc Beq nM nM Ru nM 254 36 0 0000 404 0579 2 0935
95. e poly His tagged ligand The Biacore method provided is designed for kinetic or affinity determinations but may be readily modified for other applications that use Sensor Chip NTA Biacore T100 Software Handbook Version AB Methods TS NTA kinetics Main 1 Create a new cycle type or select an existing from the list Cycle types currently in method This cycle is used in startup and sample steps Contains injection of nickel solution histidine tagged ligand sample and NTA kinetics regeneration conditioning cycle The nickel solution and histidine tagged ligand is injected over the second flow path and sample and regeneration aver both flow paths 2 Insert the required commands 3 Configure settings for each command Commands Report Points pw Yd Settings for Capture 1 aes Capture solution Nickel solution O Insert Remove From Cycle dad AC E Capture solution A0 ul min LI Contact time s DI Flow rate l min Move Li eit Flow path Second Extra wash solution Move Down T Predip Capture 2 IV Extra wash after injection with HBS EP Sample 1 SS Regeneration 1 T Stabilization period fo 9 5 11 6 Inject and Recover The example method provided for using the InjectAndRecover command is targeted to recovery for mass spectrometry and contains two assay steps The first conditions the surface by washing three times with 05 trifluoroacetic acid while the second performs the s
96. e sample plate is prepared and loaded into the instrument and a chip is docked choose Start Run to start the run 5 10 Requirements for assay specific evaluation This section describes the requirements and recommendations if assay specific evaluation is to be applied to method based runs 5 10 1 Concentration See Chapter 8 for a description of concentration evaluation e At least one assay step is required with purpose Calibration and one with purpose Sample An assay step with purpose Control Sample is also required in order to create trend plots for control samples e Assay steps Calibration Sample and Control Sample must be connected to a cycle type that includes one Sample command The three assay steps will normally be connected to the same cycle type e Samples in the Calibration step must have concentrations specified in the variable Conc At least two different concentrations are required for linear calibration curves and at least four for 4 parameter fitting e Samples in the Sample step will normally not have specified concentrations If concentrations are specified they will be ignored Biacore T100 Software Handbook 85 version AB Methods 86 5 10 2 Kinetics Affinity See Chapter 9 for a description of kinetics and affinity evaluation At least one assay step is required with purpose Sample connected to a cycle type that includes one Sample command Sample concentration must be specified in the variable
97. e test cycle see Section 4 2 1 Biacore T100 Software Handbook 43 version AB Application wizards 44 2 Assay setup Teo Surface Performance Setup E x Detection Flow path 1 X Startup v Run startup cycles Solution Dumm Number of cycles 3 Analysis Number of repetitions of the analysis cycle 20 Help lt Back Next gt Close Specify the flow path and start up cycles see Section 4 2 2 Set the number of repetitions of the analysis cycle according to the purpose of the surface performance test As a general guide the test should run for at least as many cycles as will be used normally in the assay 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 4 System preparations Check the System preparations options as required see Section 4 2 4 5 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 6 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Surface performance results When the wizard run is completed the results are opened automatically in the Evaluation Software Examine the plots of baseline and sample response against cycle number The response values should ide
98. e that the experimental conditions are comparable as far as possible To set up evaluation of the same sample series over multiple ligand densities click Multiple Rmax in the first dialog box for kinetics and affinity evaluation A panel for data subsets representing different R max values opens to the left of the curve table EB Kinetics Affinity Select Curves Create x Semple ss Temperate x Cuve red zi Include Cycle Sample Conc nM Assay step Flow ul min Contact time s Diss time s a 2 6 89 0 Samples 15 600 600 v 7 69 25 Samples 15 600 600 v 8 89 2 77 Samples 15 600 600 v 9 89 0 33 Samples 15 600 600 M 10 89 8 33 Samples 15 600 600 v 11 89 0 925 Samples 15 600 600 v 12 89 0 Samples 15 600 600 wi 13 89 0 33 Samples 15 600 600 4 14 89 2 77 Samples 15 600 600 i 15 89 0 925 Samples 15 600 600 kd RU D I Zoom lock 30 25 204 154 g t 10 x 54 of 54 10 t t t t t t t 1 200 0 200 400 600 800 1000 1200 1400 Time IV Show concentration series IV Show blank s Show average blanks s Help Multiple Armen Adjust Injection Start Back Next gt Cancel version AB Kinetics and affinity analysis Click Add to add a new data subset If you have multiple sets of the currently chosen curve type in the evaluation session e g multiple reference subtracted curves the next curve in the l
99. ed curves Controls binding Relative response for the report point binding against cycle number for control samples only for reference subtracted curves Controls stability Relative response for the report point stability against cycle number for control samples Binding levels binding Relative response for the report point binding against cycle number for samples Binding levels stability Relative response for the report point stability against cycle number for samples only for reference subtracted curves Enhancement Relative response for the report point enhance level against cycle number for enhancement injections Biacore T100 Software Handbook 99 Evaluation software general features 6 5 Custom report points 100 Report points are automatically created for all wizard and method based runs placed at strategic positions in relation to injections Choose Tools Custom Report Points to add your own report points if the default report points are not sufficient for your purposes You cannot edit the predefined report points Custom Report Points xi Custcm Report Points Assay Step Purpose 5 seconds after cycle start Sample E Add Edi Delele Help Close Click Add to add a new report point amp Add Custom Report Point i x Report point Assay Step Purpose ivi Id Cycle start vi O Startup
100. ed for all result files special items are also created for certain wizard runs see Chapter 4 6 4 1 Sensorgram An overlay plot of all sensorgrams is created and opened The sensorgrams are not aligned and are colored by assay step Biacore T100 Software Handbook Version AB Evaluation software general features 6 4 2 Plots Plots are created for most wizards if the appropriate report point is present in the results Separate plots will be created if there are multiple injections with similar report points for example baseline for capture and sample injections The Plot Settings cannot be changed but the plot can be modified using the selectors and the Tools menu see Section 7 2 Note that changing the selector settings can sometimes defeat the purpose of the plot Baseline Sample Absolute response for report point baseline against cycle number Baseline Capture Absolute response for report point capture baseline against cycle number Baseline General Absolute response for report point general baseline against cycle number Binding to reference Relative response for report point stability against cycle number for the reference flow cell Capture Relative response for report point capture level against cycle number for the capture injection Carry over Relative response for the report point co binding against cycle number for the carry over injection only for reference subtract
101. ehaviour Biacore T100 Software Handbook Version AB Thermodynamic analysis 10 Thermodynamic analysis Biacore T100 supports automated measurement of kinetics or affinity at a series of temperatures using the Thermodynamics wizard Section 4 5 4 In addition to displaying the variation of kinetic and affinity constants with temperature the evaluation software extracts standard thermodynamic parameters from the data 10 1 Background 10 1 1 Equilibrium thermodynamics For equilibrium thermodynamics the van t Hoff equation states AG RT In RTInKy D where AG is the standard free energy change R is the universal gas constant T is the absolute temperature K Kp isthe equilibrium dissociation constant Substituting in the expression AG AH TAS and rearranging gives where AH is the standard enthalpy change AS is the standard entropy change A plot of In Kp against 1 T should thus be a straight line with slope AH R and intercept on the y axis AS R This simplified relationship does not hold if the heat capacities of reagents and products differ since different amounts of energy will be required to raise the temperature by the same amount on the two sides of the reaction In such cases the plot of In Kp against 1 T is not linear and the relationship becomes Biacore T100 Software Handbook 161 version AB Thermodynamic analysis RT In Kp AH TASS AC T Tj TAC f 0 where AC
102. els this assumes that the ligand activity is unchanged between cycles in the assay it is however justified to use a local R max if there is reason to believe that the ligand activity may vary between cycles e g in a capture assay if the capture level varies between cycles Biacore T100 Software Handbook Version AB Kinetics and affinity analysis 9 2 3 Parameter significance The evaluation procedure necessarily returns values for all parameters in the fitting equations even if some parameters may not always be relevant for a particular data set The software provides two tools for determining the significance of reported parameters Check Kinetic Data and the standard error SE or T value Check Kinetic Data Clicking on the Check Kinetic Data opens a dialog that displays simulated sensorgrams based on the fitting results with the interaction rate constants k and kg varied in parallel so that the affinity constant remains unchanged If curves do not shift as values for k and kg are changed this means that the actual values are not important for the fitting and the curves do not contain kinetic information Conversely if the simulated curve shape changes as the values of k and kg are varied the fitting is dependent on the actual values and the curves do contain kinetic information This tool is only available for results obtained with the 1 1 fitting model x Compare to Modification factor M Options Sensorgram
103. emperate 37 x Cue ree zi Include Cycle Sample Conc nM Assay step Flow l min Contact time s Diss time s Ej 6 89 0 Samples 15 600 600 E 7 89 25 Samples 15 600 600 2 8 89 2 77 Samples 15 600 600 9 89 0 33 Samples 15 600 600 10 89 8 33 Samples 15 600 600 11189 0 925 Samples 15 600 600 12 89 0 Samples 15 600 600 13189 0 33 Samples 15 600 600 14 89 2 77 Samples 15 600 600 15 89 0 925 Samples 15 600 600 zi RRR KI XIX I RU Zoom lock 200 o 200 400 600 800 1000 1200 1400 Time IV Show concentration series I7 Show blank s Show average blanks s Help Multiple mak Adjust Injection Start X Back Nes Cancel A concentration series is defined by a set of curves with the same sample name analysis temperature and curve identity Select the concentration series you want to work with in the respective pull down lists Note Sample names are case sensitive so that Sample and sample belong to different concentration series Edit the sample names in the keyword table if you have unintentionally mixed upper and lower case letters If the result set contains data from more than one file curves with the same sample name temperature and curve identity are grouped together in a single concentration series Use the Include column in the table of curves to choose which curves should be included in the data s
104. er a structured sequence of settings that covers all essential aspects of the assay in question Wizard settings can be saved in templates for later use Advanced users can open wizard templates in Method Builder for more flexible assay design see Chapter 5 4 1 Wizard templates An application wizard consists of a series of dialog boxes that takes you through the steps in setting up the application Settings in the dialog boxes may be saved in wizard templates so that opening a template will present the saved settings in each dialog box Normally a wizard template is saved when all steps have been defined so that the template represents a complete assay definition including sample details if desired If a wizard sequence is closed before reaching the last step however you are given an opportunity to save the template which will then contain settings as far as they have been defined 4 1 1 Creating and editing wizard templates To create a new wizard template or edit an existing template choose File Open New Wizard Template and select the type of wizard in the dialog box Click New to create a new template or navigate to the folder where your template is stored select the template and click Open to edit an existing template The top level folder for wizard templates is defined under Tools Preferences see Section 2 4 You can navigate between subfolders under the top level in the dialog box but you cannot access files outside t
105. ermodynamics 162 10 2 Performing thermodynamic analysis 163 11 Affinity in solution 167 11 1 Conventions and background 167 11 1 1 Experimental setup 167 11 1 2 Evaluation principles 167 11 2 Requirements for affinity in solution 168 11 3 Evaluation of affinity in solution 168 A Data import and export 171 A 1 Exporting data 171 A 1 1 Export functions 171 A 2 Importing data 172 A 2 1 Control Software 172 A 2 2 Evaluation Software 175 Index 177 Biacore T100 Software Handbook Version AB Introduction 1 Introduction Biacore T100 is a high performance system for analysis of biomolecular interactions based on Biacore s surface plasmon resonance SPR technology The Control Software supplied with the system offers easy to use wizards for assay development and common applications together with flexible facilities for designing custom analysis methods using a graphical interface called Method Builder Results are evaluated in separate Evaluation Software designed for efficient and flexible evaluation with dedicated functions for common applications This Handbook describes in detail how to use the Control and Evaluation Software 1 4 System overview Instrumentation in the Biacore T100 system is described in full in the Biacore T100 Instrument Handbook Important features relevant to software operation include e Biacore T100 supports simultaneous analysis in up to four flow cells connected in series The flow cells are a
106. erwise stated 1 3 Associated documentation Biacore T100 Instrument Handbook describes the instrumentation in the Biacore T100 system with instructions for operation maintenance and troubleshooting Biacore T100 GxP Handbook describes functionality added with the optional GxP package together with some recommendations for using the system in a regulated environment Other general handbooks and documentation describing the technology are available from Biacore Information may also be found on the Internet at www biacore com 1 GxP is used as a generic abbreviation for GLP Good Laboratory Practice GMP Good Manufacturing Practice and GCP Good Clinical Practice Biacore T100 Software Handbook Version AB Control Software Biacore T100 Software Handbook 3 version AB Biacore T100 Software Handbook Version AB Control Software general features 2 Control Software general features 2 1 Operational modes Biacore T100 Control Software offers three modes of operation e Manual run provides interactive control of the instrument Operation executing commands singly as they are issued This mode is most useful for ad hoc experiments involving one or a few injections such as testing the response obtained from injection of a single sample e Application wizards provide guidance in setting up experiments for assay development and execution Separate wizards are offered for different purposes such as
107. es Click Start to start the run You will be asked to specify a result file name before the run actually starts 16 Biacore T100 Software Handbook Version AB Manual run 3 3 Controlling a manual run Control the manual run from the command buttons in the main window or the options in the Command menu Te Biacore T100 Control Software manual FrMa blr nm x EA Fie Edit View Commands Run Tools Help 8 x ug TEE 8 As AAI BO So gu i m New Cycle 3024 43 F4 P Flow 30 Cycle 1 z Curve Sensorgram Fc 1 x Hel Lock scale 33330 33320 4 33310 n c 33300 T Response 33290 33280 33270 Flow 30 Flow path 1 2 3 4 Online COMI Temperature 24 99 eC Sample compartment temperature current 25 C set 25 C Running manual run Commands are executed immediately if the instrument is idle With a few exceptions noted in the detailed descriptions below commands issued when the instrument is busy are placed at the end of a queue The queue is listed in the left hand panel with commands that have been executed in gray text and those that are pending in black text The command currently being executed is marked with a working icon Right click on a pending command for a menu with options for editing the command inserting a new command before the selected command you c
108. es the overall binding is strengthened compared with 1 1 binding This effect of often referred to as avidity A B AB AB B AB gt Note Once analyte is attached to the ligand through binding at the first site interaction at the second site does not contribute to the SPR response since there is no change in the amount of analyte at the surface For this reason the association rate constant for the second interaction is reported in units of RU s and can only be obtained in Mts if a conversion factor between RU and M is available Similarly a value for the overall affinity or avidity constant is not reported Model parameters are Obtained from ka1 Association rate constant for the first Fitted site M 1s 1 kd1 Dissociation rate constant for the first Fitted site s ka2 Association rate constant for the Fitted second site RU s kd2 Dissociation rate constant for the Fitted second site s Rmax Analyte binding capacity of the Fitted surface RU Conc Analyte concentration M Provided as input tc Flow rate independent component of Fitted the mass transfer constant f Flow rate pl min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in Fitted the sample Biacore T100 Software Handbook Version AB The following values are presente
109. esult file and click Affinity in Solution on the toolbar The first step displays the calibration curve for measurement of free B Biacore T100 Software Handbook Version AB Affinity in solution A Affinity in Solution Step 1 H557 thrombin 2 xi m Calibration Curve Settings Sensorgram Fc 2 z Report point tabiiy v Response type Relative response Fitting function 4 parameter 7 Calibration Curve and Sample Selection sone EST Catsien cur Eaman Calibration Curve Information Calibration Curve Cycle Conc B Response Calc Conc B cv RU nM Ru nM 500 4 4 0 0020 21 9049 0 0026 16 0 0020 21 3073 0 0025 4 Avg 21 6061 0 0026 N A 400 4 5 0 0039 29 1999 0 0040 350 7 17 0 0039 27 4792 0 0037 300 4 Avg 28 3395 0 0039 NJA 8 H i 5 2504 6 0 0078 40 8066 0 0070 a e 200 4 18 0 0078 39 8743 0 0067 Avg 40 3405 0 0068 N A 150 4 0 0156 61 5417 0 0145 iu Parameters 50 4 Rhi 673 604 Rlo 8 901 Al 0 923 A2 0 521 0 t t t t t t t t t 1 Ch 6 065 0 5 0 05 1 15 2 25 3 35 4 45 Concentration B nM Help Back Nest Cancel Choose the sensorgram report point response type and fitting function from the pull down lists at the top of the dialog See Section 8 2 1 for more details of these choices If you have run multiple sample series in the experiment choose the sample to evaluate in the Sampl
110. esults are opened automatically in the Evaluation Software Predefined plots Section 6 4 are created for each sample 4 5 2 Concentration analysis The concentration analysis wizard helps you to set up an assay for determining analyte concentration in samples with the help of a calibration curve using known concentrations Control samples may be included at intervals to monitor the stability of the assay 1 Injection sequence Choose the injection sequence for the concentration analysis see Section 4 2 1 2 Assay setup Specify the flow path and start up cycles see Section 4 2 2 Start up cycles are recommended for concentration assays to ensure that the initial response drift that may occur with a new chip does not interfere with the first measurements 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 Biacore T100 Software Handbook 49 version AB Application wizards 50 The sample injection for concentration analysis can be extended to include a mixing function whereby sample is mixed with a specified proportion of a second fixed solution T Concentration Analysis Injection Parameters xj Sample Contact time 120 s Flow rate fio ul min v Mix with Detecting molecule Fraction 30 ofthe mix solution m Regeneration Solution name Regeneration T High viscosity solution Contact time 30 s Flow rate 30 ul min Stabilization
111. et to be evaluated You can select several curves and use the right click menu to exclude or include multiple curves in one operation By default all curves for the sample are included sensorgrams for non zero concentrations are shown in color and those for blanks zero concentrations in light gray The sensorgrams are adjusted to zero at the start of the sample injection on both the response and time axes The average Biacore T100 Software Handbook Version AB Kinetics and affinity analysis of the blank sensorgrams will be automatically subtracted from the other curves when you proceed to the next step If you do not want to perform blank subtraction exclude the zero concentration sensorgrams from the data set You can also choose to use blanks from other concentration series for blank subtraction these are listed at the bottom of the table and are excluded by default Only blank sensorgrams with the same contact and dissociation times as the samples are used For runs that use captured ligand the ligand is listed in the table Make sure you only include curves that are derived from the same ligand The three check boxes below the sensorgram panel control the type of curves shown in the display You can use these check boxes to examine the sample and blank curves without interference from each other and to show the average blank that will be used for subtraction Bear in mind however that these boxes control the display only and d
112. et up a pair wise epitope mapping experiment Click Import to import the sample data from an external file Import of sample information must be enabled in Tools Preferences to use this function See Appendix A for details of import functions and file formats Click Control Samples to enter control samples for the run Teo Binding Analysis Control Samples M Biacore T100 Software Handbook Version AB Application wizards Specify the details of control samples and the frequency with which they should be run Check the Repeat control samples every box and enter a number of cycles to repeat the control samples at intervals during the run The controls will then be run at the beginning of the assay and at the specified interval If this box is not checked the control samples will be run once at the beginning of the assay You can uncheck the Run control samples box to skip control samples without deleting the details from the method 5 System preparations Check the System preparations options as required see Section 4 2 4 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Binding analysis results When the wizard run is completed the r
113. etection Flow path 2 1 b Startup IV Run startup cycles Solution Dummy sample Number of cycles 3 M Temperatures Analysis temperatures Sample compartment temperature Temperature js m d punc 10 x lt Back Next gt Close Specify the flow path and start up cycles see Section 4 2 2 Start up cycles are recommended for thermodynamics experiments to ensure that the initial response drift that may occur with a new chip does not interfere with the first measurements Start up cycles will be run at each temperature Enter the temperatures at which the measurements are to be performed For most purposes 5 7 temperatures will be adequate fewer points make the determination of thermodynamic parameters uncertain while more points increase the run time Distribute the points evenly over the widest temperature interval that the ligand and analyte tolerate Start from the lowest temperature to minimize the time needed for temperature equilibration between measurements increasing the analysis temperature takes less time than decreasing it The system will wait for a stable temperature between each determination As an additional control you may want to include a replicate of the first temperature at the end of the run Note For robust determination of standard thermodynamic parameters AG AH and AS arrange the analysis temperatures so that 25 C is in the middle of the
114. ethods stored in other locations Check Show importable wizard templates to display wizard templates that can be opened in Method Builder Opening a wizard template imports all wizard settings into a method and allows you to add functionality that is not supported in the wizard Templates from all wizards except immobilization can be imported into Method Builder Biacore T100 Software Handbook 63 version AB Methods 5 2 Method structure 64 Methods are handled in Method Builder in a series of sections representing different aspects of the method definition Overview The Overview screen summarizes the method definition Use this information as an aid in checking that the method is correctly built General settings Here you define general parameters such as the concentration unit for samples sample compartment temperature data collection rate detection mode and buffer names Assay steps An assay step represents a specific function in the assay defined in terms of what the step is intended to achieve Assay steps may for example be start up operations solvent correction sample analysis or control sample analysis Steps can be run singly or repeated within the context of other steps for example start up operations are typically performed once at the start of a run while control samples may be repeated at intervals during the sample analysis Analysis temperature and buffer can be set individually for each assay step Cyc
115. expressed as a T value which is obtained by dividing the value of the parameter by the standard error A high T value corresponds to a low standard error As a general guideline parameters with a T value greater than about 10 should be regarded as significant The choice of whether to display parameter significance as standard error or T value is made on the Fit tab of the Tools Preferences dialog The significance of a parameter is a measure of how much a change in the parameter value affects the closeness of fit A parameter with low significance can have a wide range of values without affecting the fit Typically but not always parameters with a low significance have unreasonable values for example typical values for the mass transfer constant for proteins are around 10 RU M s but evaluation of data with no mass transfer limitation might return a value of 1012 or higher Similarly rate constants that lack significance are often assigned values outside the reasonable range for biomolecular interactions or outside the range that can be measured with Biacore Notes The standard error and the Check Kinetic Data tool assess parameter significance in different ways even if the results of the assessment may sometimes be related Check Kinetic Data tests the contribution of a group of parameters rate constants for the interaction and mass transport processes to the closeness of fit by examining the results of correlated changes whereas
116. ext in either ASCII or Unicode format conforming to the following specification Biacore T100 Software Handbook 173 version AB Affinity in solution 174 e Two lines in the file specify the microplate and reagent rack settings in the format Rackl microplate specification Rack2 reagent rack specification Specifications are not case sensitive but microplate and reagent rack specifications must be given otherwise exactly as they appear in the selection lists in the Rack Positions dialog If either specification is invalid the corresponding definition will not be imported The position of these two lines in the file does not matter e One line specifies the headers for table columns to be imported separated by tabs The headers should correspond to the column headers as they appear in the Rack Positions table with the exception of the Volume column in the table which can be omitted from the import file and is ignored if it is present This line may nor be preceded by any line other than the microplate and reagent rack specifications e A set of lines hold the content of the table columns separated by tabs When import is requested the contents of each table line in the import file are matched as far as possible to the contents of the Rack Positions table with the exception of the Position and Volume column For matched rows the Position in the table is replaced by the value in the Position column from the import file
117. f solvent correction solution over the surface at a flow rate of 30 pl min A solvent correction cycle should contain 4 8 Solvent correction commands for the different solvent concentrations used to construct the correction curve see Section 6 7 Solvent correction commands will be correctly evaluated only when they are used in cycle types that are connected to assay steps with purpose Solvent correction 5 6 2 Variables Parameters for several of the commands in a cycle definition may be set as variables Values for variables are entered in either the Sample and Assay Setup or the Setup Run step Sections 5 7 and 5 9 2 and determine the number of cycles that will be performed in the run Variables fall into two broad classes e Method variables such as sample name or contact time that control the way the cycle will be performed Parameters that are not set as variables are defined in the main command panel Biacore T100 Software Handbook 77 version AB Methods 78 e Evaluation variables such as concentration or analyte molecular weight that are used in evaluation of the data Some evaluation variables are required for correct functioning of application specific evaluation procedures for example kinetic evaluation requires a variable called Conc which holds the analyte concentration These are selected from a predefined list Other evaluation variables may be freely defined by the user to hold information that is relevant to the
118. g molecule at the beginning of a cycle The injected solution contact time and flow rate can be set as variables Carry over control command This command injects a 30 second pulse of buffer over the surface at a flow rate of 40 pl min in order to check that there is no carry over of analyte or other material from an injection earlier in the cycle The injection is suitably placed at the end of the cycle and can be used in a conditional context see the If then command to perform additional buffer injections or regeneration steps if carry over is detected A plot of the binding response from a carry over injection against cycle number is created automatically for quality control purposes in the evaluation software see Section 6 4 If then command This command allows construction of conditional methods where commands are executed or skipped depending on the outcome of certain conditions The illustration below shows a cycle which will perform an additional regeneration if the relative response after the first regeneration exceeds a specified value Commands Report Points Regeneration Condition If For Report Point Flow cell Comparison Insert Command Relative Response Y regeneration v reference21 Greater Than gt Remove From Cycle Constant E 10 Move Up T Use additional condition Move Down If statement is true then Command Sequence E If condition True 4 If statement is false
119. g window Choose the Wizard Template or Method options to display the wizard or method definition for the run You can edit the definition and save it as a new wizard template or method You cannot however change the original definition that is saved together with the result file Notebook opens a notebook window where details of the run may be recorded The notebook is only available during a run or for a completed result file For immobilization and regeneration scouting wizard runs the Wizard Results option opens a window showing the results of the run All other runs are evaluated in the Evaluation Software Sensorgram Markers controls display of report point and event markers and labels in the sensorgram window 2 3 5 Run menu The options in the Run menu are used to start the different types of runs see Chapter 3 and Sections 4 1 2 and 5 9 7 Biacore T100 Software Handbook 11 version AB Control Software general features Tools Prime Shutdown Biacore T100 Evaluation Software Standb Stop Standby Eject Rack Rack Illumination amp Set Temperature Preferences More Tools 12 2 3 6 Tools menu Options in the Tools menu control instrument operations outside the context of runs Prime flushes the flow system with fresh buffer There is an option to include Prime at the beginning of each wizard or method based run Use the menu option when you want to flush t
120. grams further experimental efforts to reduce the heterogeneity are recommended where possible A B1 AB1 A B2 AB2 Note The model is limited to two ligands because the fitting algorithm tends to become unstable with more components and three or more ligand species cannot be reliably resolved Model parameters are Obtained from kal Association rate constant for the first Fitted ka2 and second ligands M7 s kd1 Dissociation rate constant for the first Fitted kd2 and second ligands s Biacore T100 Software Handbook 149 Kinetics and affinity analysis Rmax1 Analyte binding capacity of the first Fitted ligand RU Rmax2 Analyte binding capacity of the Fitted second ligand RU Conc Analyte concentration M Provided as input Tc Flow rate independent component of Fitted the mass transfer constant F Flow rate pl min Provided as input TOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in Fitted the sample The following values are presented in the report Calculated as ka1 Association rate constant for the first kal ka2 and second ligands M ts 1 ka2 kd1 Dissociation rate constant for the first kd1 kd2 and second ligands s kd2 KD1 Equilibrium dissociation constants kd1 ka1 KD2 M kd2 ka2 Rmax1 An
121. has a relative viscosity higher than about 3 corresponding to about 3596 glycerol or 40 ethylene glycol at 20 C This will adjust the injection procedure to ensure correct handling of viscous solutions and will limit the maximum contact time that can be specified Wait Inserts a Wait command in the queue causing the instrument operation to pause for the specified time period Buffer continues to flow over the sensor surface during the Wait period and data collection continues Eject Rack Tray Ejects the rack tray so that you can load more samples Do not change the type of microplate or reagent rack on the tray This command is inserted immediately after the command currently under execution rather than at the end of the queue so that the rack tray will be ejected as soon as the current command is completed If you want to place the command later in the queue use the right click menu in the queue panel to insert the command at the appropriate place Biacore T100 Software Handbook Version AB Manual run WARNING The rack tray automatically moves into the instrument 60 seconds after it has been ejected A timer in the dialog indicates when the rack tray will be automatically moved into the instrument New Cycle Starts a new cycle You can choose a new flow path and reference subtraction setting for the new cycle independently of the setting in the current cycle Stop command Stops the command currently be
122. have not been used and shows Blank for flow cells that have been prepared as a blank reference surface by activation and deactivation The text Incomplete results indicates that the immobilization run was interrupted by for instance user intervention or power failure before it could be completed Wizard Results Ctr Sensorgram Markers 10 xi Chip id Chip lot no Firstuse date 050915 03nn 12004 2005 09 15 Chip IFC type CM4 IFC105 Flow cell ur ai nmal Ro Ligand Result file Fe 1 i 2006 02 09 i 2438 6 i Anti b2m C BI4 Users Immobilization 060209 1 blr Fc 2 2006 02 09 Blank C BIA Users Immobilization 60209 1 blr Fe 3 2006 02 14 5532 2 Anti b2m C BIA Users Immobilization 0602 1 4 1 blr Fc 4 2006 02 14 Incomplete results C BIA Users Immobilization 060214 1 blr Help Properties for the sensor chip used in a currently open run may be found under File Properties Section 2 3 2 Title sets a title in the sensorgram window The default title is the assay step name Scale sets the scale of the sensorgram window ERN 09 X Axis Scale Y Axis Scale Auto scale v Auto scale Min o Min Max 1800 Max 170000 Help Cancel If you set Auto scale the scale will be adjusted if necessary to accommodate the full data range of the currently displayed cycle During a run the scale is adjusted at intervals as more data is collected Check the Lock scale box in the top right cor
123. he same order of magnitude as the measured binding response Biacore T100 Software Handbook Version AB Evaluation software general features Solvent correction should not be applied in situations that do not meet all three of these criteria Attempts to use solvent correction in other circumstances may introduce errors that are larger than the solvent effects that the procedure is intended to correct 6 7 3 How solvent correction works Solvent correction factors are determined by injecting a series of blank samples containing a range of solvent concentrations and plotting the difference in relative response between the active and reference surfaces as a function of the relative response on the reference surface Each sample measurement is then corrected by a factor obtained by measuring the relative response on the reference surface and reading the corresponding difference between active and reference surfaces from the correction curve see illustration Ref subtracted Ref subtracted ee a RU Corrected Reference yas The principle of solvent correction 1 The sensorgram from the reference flow cell shows a bulk displacement 150 RU in the illustration during sample injection because the sample and running buffer are not exactly matched 2 From the solvent correction curve a displacement of 150 RU in the reference sensorgram corresponds to a solvent error of 5 RU in the reference subtracted
124. he scale for the display You can also access this function by double clicking on either the x or y axis in the display Biacore T100 Software Handbook 97 version AB Evaluation software general features CINE xi x Scale Y Scale IV Auto v Auto Logarithmic Logarithmic Min D Min o May oo May 5200 Cancel Uncheck Auto and enter minimum and maximum values to specify a scale Copy graph Copies the current display as it appears on the screen to the Windows clipboard as a graphic object from where it can be pasted into third party software such as word processing or presentation programs Export curves Exports the curves in the current display to a tab separated text file for import to third party software Complete data is exported regardless of the scale setting of the screen display Unzoom Restores the previous zoom setting Gridlines Shows or hides major and minor gridlines in the display window Legend Shows or hides a legend for the display window Choose the legend placing from the dialog box The legend content varies with the type and settings for the display window but in principle identifies the different curves or sets of points in the window 6 4 Predefined evaluation items 98 When a result file is opened a number of evaluation items are created automatically if the results contain the appropriate cycles and report points This section describes the items creat
125. he system at other times e g before a manual run Shutdown starts the procedure for shutting down the instrument for long periods of time more than 4 days The procedure displays necessary instructions on the screen Details of the shutdown procedure are given in the Biacore T100 Instrument Handbook Standby puts the instrument in standby mode which maintains a low buffer or water flow through the flow system for up to 4 days Leaving the instrument in standby mode when not in use is generally recommended The instrument is automatically put in standby mode at the end of a run Use the menu option if standby has been stopped and you want to restart it Stop Standby stops standby mode Eject Rack ejects the rack tray from the sample compartment The rack may be ejected during setup for wizard and method based runs and at any time during a manual run Use the menu command when you want to eject the rack at any other time WARNING The rack tray automatically moves into the instrument 60 seconds after it has been ejected A timer in the dialog indicates when the rack tray will be automatically moved into the instrument Rack Illumination switches the blue illumination in the sample compartment on or off The illumination helps you to see in the sample compartment but does not otherwise affect instrument function Insert Chip and Eject Chip are used for docking and undocking the sensor chip respectively More details are given i
126. he top level folder from within the dialog box Click Browse to navigate freely in the computer file structure and open wizard templates stored in other locations Note The Open New Wizard Template dialog box only lists templates of the selected type but the Browse dialog may list all types Template types are identified by the file extension which may Biacore T100 Software Handbook 21 version AB Application wizards or may not be displayed according to your Windows Explorer settings see Section 2 4 1 4 1 2 Running wizards When you start a run based on a wizard template you step through the dialog boxes in the template and may change settings if desired 22 Biacore T100 Software Handbook Version AB Application wizards 4 2 Common wizard components Several dialogs are common to a number of wizards with equivalent functions and only minor differences if any These steps are described in this section Any wizard specific variations in these common components are described in the section on the appropriate wizard below 4 2 1 Injection sequence This dialog determines the sequence of injections in the wizard analysis cycle Some injections are not supported in certain wizards e g the kinetics wizard does not support enhancement injections Te Concentration Analysis Injection Sequence i xj CAPTURE V Capture SAMPLE Sample Enhancement REGENERATION 1 v Regeneration E Help Back Next gt Clos
127. he unit for entering sample concentrations The unit must be specified here and cannot be changed at any other step in the assay definition At start Data collection rate Choose between 1 and 10 Hz for data collection The higher setting will provide better resolution for kinetic analysis of fast interaction processes but will result in larger result files At start Sample compartment temperature This is the temperature in the sample compartment not the temperature at the flow cell which is set for each assay step Check the Vary with analysis temperature box to set the rack temperature automatically to the same value as the analysis temperature After run Analysis temperature Check this option and enter a temperature to set the analysis temperature when the run is completed The rack temperature will also be reset if the Vary with analysis temperature box is checked This setting provides automated control of the chip and detector environment after the completion of a run for example in preparation for another run at a different temperature Detection Choose between Single detection in one flow cell according to the chosen flow path Dual detection in a flow cell pair according to the Biacore T100 Software Handbook Version AB 5 5 Assay steps Methods chosen flow path and Multi detection in all four flow cells This setting affects the choice of flow path that can be made for each injection command in the cycle type
128. hether variable parameter values are specified in the method or at run time You may choose to specify all values in the method all at run time or a mixture of the two Values that are specified in the method are saved with the method and cannot be changed at run time You can change the level at which values are specified without changing the cycle type definition so that the same cycle type can be used for different assay steps with different sets of run time variables An example of this may be found in the predefined method for affinity in solution Section 5 11 1 Variables are configured independently for the different assay steps even if the assay steps use the same cycle type If you choose to specify all variables in the method the values are entered in this screen For each assay step one row of variable values represents one analysis cycle the cycle may be repeated if the Repeat property is set in the Assay Step screen Each row in the variables table corresponds to a cycle in the run A new empty row marked with an asterisk is created automatically at the bottom of the table as soon as data is entered Columns in the table correspond to variables for the cycle type used in the assay step and are grouped according to commands in the cycle type definition Use the right mouse button in the variables table to access functions for copying and pasting cell contents and for inserting and removing rows When all variables are specif
129. hoose the command to insert from a dialog box e deleting the command You can also use the right click menu to copy selected command or commands and paste them elsewhere in the queue The Copy function works with both completed and pending commands Biacore T100 Software Handbook 17 version AB Manual run 18 Flow rate Sets the flow rate to a new value Flow path Changes the flow path During a cycle you can only select a flow path within a range allowed by the setting chosen when the cycle was started for example if the current setting is Flow path 1 2 you cannot extend it to Flow path 1 2 3 4 Sample inject Injects sample Choose the position from which the sample will be taken and specify a contact time Positions that can be chosen are determined by the rack settings in the manual run start up dialog Make sure that the chosen position contains enough sample for the injection The required volume for the specified contact time is indicated in the dialog box Regeneration inject Injects regeneration solution Choose the position from which the solution will be taken and specify a contact time Positions that can be chosen are determined by the rack settings in the manual run start up dialog Make sure that the chosen position contains enough solution for the injection The required volume for the specified contact time is indicated in the dialog box Check High viscosity solution if your regeneration solution
130. hoose Copy from the right click menu The selected contents will be copied in tab separated text format to the Windows clipboard and can be pasted from there into other programs All selected cells will be copied including header cells and filter settings Biacore T100 Software Handbook 121 version AB Data presentation tools 122 Biacore T100 Software Handbook Version AB Concentration analysis 8 Concentration analysis Biacore T100 software supports evaluation of concentration analysis using both linear and non linear calibration curves determined by measurement of known samples 8 1 Requirements for concentration evaluation In order to evaluate concentration analysis from a Biacore T100 run the run must include at least one calibration curve and unknown sample injections must have the appropriate properties and keywords For wizard based runs the conditions are met automatically when the Concentration Analysis wizard is used For method based runs the method must be constructed as described in Section 5 10 1 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command name cannot be edited in the keyword table Refer to Chapter 5 for details of how to construct methods in Method Builder Evaluation of unknown samples is based on the nearest preceding calibration curve If there is no preceding calibration curve in the run the nearest following cur
131. ied at run time variables are handled in the same way in the Setup Run step To specify that some variables are specified in the method and others at run time check the appropriate option and then distribute the variables Biacore T100 Software Handbook 81 version AB Methods 5 8 Verification 5 9 Setup Run 82 as required between the method and run time lists This mode can be used to hide variables at run time that are not relevant for the assay step Fill in values for variables that are specified in the method those specified at run time are filled in the Setup Run step Note that in this mode only one set of variable values can be specified in the method the number of cycles is determined by the number of rows of variable values in Setup Run Depending on how the method is defined there may be variable tables for several assay steps Variable handling must be defined for all steps before the method will pass verification This step checks that the method is correctly and completely defined A method that does not pass the verification step can be saved but cannot be run Verification may fail because parameters are missing e g variables that are specified in the method have not been assigned values or because the method construction is invalid e g an assay step is not connected to a cycle type 5 9 1 Detection Set the flow path for the method in the Detection dialog T Method Builder Detection xi Detection
132. immediately after the currently marked command or at the end of the cycle definition if no command is marked Use the Move up and Move down buttons to change the position of the command in the cycle Commands are executed from top to bottom in the cycle definition General command properties Common features of several commands are check boxes for Predip Extra Wash After Injection and Stabilization period Predip Check this box to dip the needle in a separate position before aspirating the solution to be injected The predip position will normally contain the same solution as is injected so that the needle is rinsed briefly to minimize carry over effects The same predip position is used for all cycles Extra Wash After Injection Check this box and specify a wash solution to perform an extra wash of the flow system after the injection The flow system is washed automatically with buffer after each injection but an extra wash with a different solution can be included if required This wash solution does not pass over the sensor surface Biacore T100 Software Handbook Version AB Methods Stabilization period Check this box and specify a time in seconds to introduce a delay before the next command is started This can sometimes be necessary for example after regeneration steps to allow the response to stabilize before performing the next injection Capture command This command is intended for injection of ligand over a capturin
133. ing The regeneration scouting wizard guides you through the process of finding suitable regeneration conditions for your sensor surface The principles of regeneration scouting are described in the Biacore Sensor Surface Handbook Briefly regeneration scouting is performed by testing repeated cycles of analyte injection and regeneration over a range of regeneration conditions and assessing the results on the basis of trends in analyte response and baseline levels The analyte concentration should be relatively high for best results The analyte response reflects the binding capacity ligand activity of the surface while the baseline level indicates the extent of regeneration Each condition should be tested for at least 3 cycles in sequence recommended number 5 in order to detect trends in the regeneration behaviour with the given condition When testing multiple conditions start with the mildest conditions to minimize the risk of losing ligand activity at the beginning of the scouting series 1 Injection sequence Choose the injection sequence for the regeneration scouting see Section 4 2 1 One sample injection and one or two regeneration injections are required Most sensor surfaces can be adequately regenerated with a single injection but some situations may benefit from using multiple injections 2 Setup Specify the flow path Tis Regeneration Scouting Setup x Detection Flow path Startup 7 One startu
134. ing executed The icon changes to 3 show the command that will be stopped or is gray if the current command cannot be stopped e g it is not possible to stop an Eject Rack Tray command Stop Run Finishes the run ul Pause Run Pauses the run until a Resume Run command is issued Buffer continues to flow over the sensor surface while the run is paused lE Resume Run Resumes a run that is paused E Add report point B Adds a report point to the sensorgram Help Displays help for the manual run Biacore T100 Software Handbook 19 version AB Manual run 3 4 Ending a manual run To end a manual run 20 J Issue a Stop Run command The command will normally be placed at the end of the queue If you want to stop the run before the queue is completed use the right click menu in the queue panel to delete commands from the queue or to insert the Stop Run command in the appropriate position Choose Tools Eject Rack to eject the rack tray and remove your samples and reagents Choose Tools Eject Chip to undock the chip if desired Biacore T100 Software Handbook Version AB Application wizards 4 Application wizards Application wizards guide you through the procedure of setting up common applications with recommendations and settings based on Biacore s expertise in the field of SPR based interaction studies Wizards are an ideal starting point for inexperienced or infrequent users since they off
135. interaction or as an equation defining response as a function of time Interaction models are described in Section 9 4 1 and equation models in Section 9 4 2 Predefined models cannot be edited or removed If you want to modify a predefined model create a new model using the predefined model as a template Biacore T100 Software Handbook 153 version AB Kinetics and affinity analysis 9 4 1 Interaction models for kinetics The reaction scheme for an interaction model supports up to 5 component reactions Follow the steps below to define a new model or edit an existing definition ER Eit mosen xi Model Name Description Analyte A binds to ligand B Includes terms for mass transfer of analyte to the surface Reaction React 1 React2 React3 k forward k backward E uM iE oe B E SEE BNN 3 E E oo E aE 13 E E Interaction Parameters Reactants B Category Analyte s Concentration oc Injection s tOn for Masstransfer feria Molecular weight a I No of blocked stes New Delete Bulk And Drift Response RU From s To s IV Refractive inder RI bon for Refractive index 2 Ec SS T Drit in RU nm es d 1 On the Interaction tab click New to add new reactants For each reactant choose whether it is analyte ligand or complex see below and enter an identifier for the reactant Enter parameter names or e
136. is can result in contact times that are longer than requested for example at 1 pl min a requested contact time of 90 s requiring 1 5 pl solution will result in an actual contact time of 120 s solution volume rounded up to 2 pl Stabilization time after injection This function is available after a capture injection and after the last injection in the sequence For capture injections a stabilization time can be useful if a fraction of the ligand dissociates rapidly Including a stabilization time to allow for such dissociation can help to improve reproducibility A stabilization time may be used after the last injection instead of regeneration for systems where analyte dissociates rapidly and completely from the surface Exposure of the surface to regeneration solution can often lead to transient changes in the baseline Inclusion of a stabilization time after regeneration helps to ensure a stable baseline for the next cycle Sample injection Normally the injected sample solution is specified in a separate sample table Some wizards e g Surface Performance use only a single sample solution that is specified together with the other injection parameters in this dialog box Regeneration The parameters for regeneration include a check box for High viscosity solution Check this box if the regeneration solution has a relative viscosity higher than about 3 corresponding to about 3596 glycerol or 40 ethylene glycol at 20 C This wi
137. is option controls whether the samples and reagents will be placed in the reagent rack or the sample microplate If Auto is chosen placement is decided on the basis of number and volume of solutions in the category Vial size Use this option to determine the vial size for reagents If Auto is chosen placement is decided on the basis of the volume of solution Pooling This option allows you to combine solutions with the same name into one position or to split combined solutions into separate positions for each cycle Choose Yes to pool solutions if suitable vial positions are available or No if you always want separate positions for each cycle Choose Auto to Choose Auto to set the pooling according to the type of region Sort by Solutions within a category may be sorted by one or two parameters Use the Move up and Move down buttons to change the order in which regions are listed Regions are placed in the specified rack or plate in the order listed so that changing the order of the table can change the automatic positioning of samples and reagents Save Wizard Template Save Wizard Template As Saves the wizard template with either the same or a different file name The corresponding function is also available for methods Automatic Position Import This option imports positioning information from an external source The option is only available if Enable automatic rack positions import is checked in Tools Preferences Choosing the op
138. is the heat capacity change under standard conditions and T is the reference temperature 25 C 298 15 K for standard conditions A value for the standard heat capacity change AC can thus be obtained in addition to AH and AS from non linear fitting of the data to this extended equation 10 1 2 Transition state thermodynamics Transition state theory holds that the equilibrium constant for formation of the transition state in a reaction can be related to the rate constant for the overall reaction by the Eyring equation 2 where Kt is the equilibrium constant for formation of the transition state for the forward or back reaction k is the kinetic rate constant for the interaction in the corresponding direction k or kg h is Planck s constant kg is Boltzmann s constant Applying a similar rearrangement of the thermodynamic equations for the transition state gives ln kh AH P AS kT RT R so that the thermodynamic transition state constants for the forward and backward reactions can be obtained from plots of In k T and In kg T respectively against 1 T Note that the Eyring equation does not have a corresponding non linear form that takes account of the heat capacity change for transition state formation Non linear fitting to obtain values for AC can only be applied to equilibrium thermodynamic analysis 162 Biacore T100 Software Handbook Version AB Thermodynamic analysis 10 2 Performing thermodynami
139. is used in startup calibration and sample steps S Contains injection of sample and regeneration Assay Steps Cycle Types Sample amp 2 Insert the required commands 3 Configure settings for each command EE pem id owe Settings for Sample 1 Low sample consumption T Method Variables Evaluation Variables Insert Command Predefined variables tion Is variable Remove From Cycle Name Value type Contact time EE Z ConcB calibr Numeric Move Up V ConcA variable Numeric pcm C ConcB fx Numeric Move Down Flow rate HO ul min Sample 1 Regeneration 1 Flow path Ban Z User defined variables T Predo Name Value type T Mix with Fraction D of mix solution T Stabilization period after mi t i Add Delete T Extra wash after injecionwih pT Evaluation purpose I Stabilization period o is Affinity in solution gt 4 Biacore T100 Software Handbook 87 version AB Methods The Sample and Assay Setup is different for the two assay steps so that only the relevant variables are entered at run time for each assay step 5 11 2 Heterogeneous analyte kinetics This method is a straightforward kinetics determination with evaluation variables included for the evaluation purpose Kinetics heterogeneous analyte providing separate concentration and molecular weight variables for two analytes Ta Kinetics heterogeneous analyte Main This cycle is use
140. ist is assigned to the new data subset If there are no more curves of the same type available the new set will be a copy of the most recently created subset You can add up to five data subsets representing five ligand densities The same sample name and analysis temperature apply to all subsets Click on a subset to manage the contents of the subset You cannot mix subsets that use different samples or different temperatures Caution Make sure that the same curve numbers are not assigned to more than one data subset If a curve is assigned to two data sets the software will try to evaluate the same curve with two different Rmax values and the fitting may be distorted Do not evaluate multiple copies of the same subset for multiple ligand densities Subsets that are duplicated will be weighted more than those that are not duplicated in the fitting procedure Do not attempt to use subsets for any purpose other than multiple ligand densities The data will be evaluated in terms of multiple ligand densities regardless of how you have assigned curves to the subsets The subsets will be evaluated together with rate constants that are global for the whole data set and Rmax values that are global within each subset but can differ between subsets Note Evaluation with a global Rmax parameter within subsets is achieved by using a control parameter in which is set to 1 for the mth subset and 0 for all others Rmax is then fitted as Rmax in
141. ith the same concentration of component B mixed with different concentrations of component A are required Biacore T100 Software Handbook Version AB Methods 5 10 5 Other requirements Application of solvent correction see Section 6 7 requires an assay step with purpose Solvent Correction connected to a cycle type that includes at least four Solvent Correction commands for different solvent concentrations 5 11 Method examples A selection of predefined methods covering common applications is provided in the Biacore Methods folder see Section 5 1 Use these methods either directly or as starting points for your own method development This section describes the essential features in each method that are not supported in wizards Refer to these methods as guidelines in constructing your own methods that exploit similar features 5 11 1 Affinity in solution This method is designed for measurement of affinity in solution as described in Chapter 11 The method includes a Calibration assay step for measurement of component B and a Sample step for measurement of mixtures of components A and B Both these assay steps are connected to the same cycle type Predefined evaluation variables are included for the evaluation purpose Affinity in solution Te Affinity in solution Main De 1 Create a new cycle type or select an existing from the list Cycle type description General Settings Cycle types currently in method This cycle
142. kes or other disturbances x Curves Edit Cycles Curve Conc nM Assay step Flow ul min Contact time s Diss time s 2 7 Feel 25 Samples 15 600 600 8 Feti 2 7 Samples 15 600 600 9 Feel 0 33 Samples 15 600 600 10 ceo 8 33 Samples 15 600 600 11 Feel 0 825 Samples 15 600 600 18 Fai 0 33 Samples 15 600 600 14 Fe 4 1 2 77 Samples 15 600 600 15 Fai 0 825 Samples 15 600 600 16 Fe 4 1 H 25 Samples H 15 600 600 17 Feci 3 33 Samples 15 600 600 x Remove Selection Undo RU Blank Subtracted Sensorgrams 7 Zoom lock RRR K KI amp KI X XJ Response 200 o 200 400 600 800 1000 1200 1400 lt Back 1c Affiity gt io Kinetics gt Cancel Biacore T100 Software Handbook Version AB Kinetics and affinity analysis To delete a selected region from all curves drag with the right mouse button over the region to be deleted and click Remove Selection Click Undo to restore the deleted data If you want to delete a region from only selected curves remove the checkmark from the Edit column in the table for the curves that are to be left unchanged All curves are selected by default and are shown in dark color Curves that are not selected for editing are shown in light color Note that all curves will be evaluated whether they are selected for editing or not removing the Edit checkmark does not exclude a curve from the data set for evaluation Click
143. l likelihood meaningless if the data is obtained from a mixture of different models Check the rows for data that you want to use in the thermodynamic evaluation Use the Check All and Uncheck All as quick options to select and deselect the whole list Note If you use data from fitting models that include multiple rate or affinity constants be sure to select the correct rows so that equivalent constants are included from each fit In some cases it may be necessary to examine the kinetic or affinity evaluation items to determine which constants belong together Biacore T100 Software Handbook 163 version AB Thermodynamic analysis Click Next when you have selected the data to be included The results are displayed first as plots of affinity and rate constants against temperature E Thermodynamics Overview Create x Thermodynamic Overview M KD Evaluation Temperature C Description ka 1 Ms kd 128 87 bani iod 4 2 296E 5 383 too b2micro 12 12 4 002E 5 6 61 1 b2micro 19 19 5 070E 5 141 8e 9 4 b2micro 26 26 652945 287 v b2micro 33 3 636E55 6664 amp 6e 9 b2micro 40 40 1 657E 6 1864 i 4e 9 4 Y Y 2e 8 4 X v 04 t t t t t t 34 t 1 0 5 10 15 20 25 30 535 40 45 li Temperature C p jen 1Ms ka Tis kd 1 826 5 0 02 v 18e6 Y 1426 0 015 1 2e6 0 01 1e6 4 v S s0000 z hd 5e 3 600000 z v 400000 4 v 7 Y v 200004 Y 0 4 1
144. le types Cycle types define the details of how assay steps will be performed in terms of sample and reagent injections Each assay step is linked to one cycle type but the same cycle type can be used in multiple assay steps For example sample and control sample analysis are two assay steps that will typically use the same cycle type ensuring that controls are analysed in exactly the same way as samples Parameters for injections in a cycle type definition may be variable so that they can be assigned a series of different values when the method is used Sample names will typically be variable The number of values for variable parameters together with assay step repetition determines the number of cycles that will be run Report points can also be defined for each cycle type Sample and assay setup This section determines how values for variable parameters are specified You can choose whether values are specified in the method or at run time this can be used to restrict the number of parameters that have to be entered when the method is run while at the same time maintaining flexibility for method development purposes Biacore T100 Software Handbook Version AB Methods Verification Once the method has been defined in full this section verifies that all aspects are consistent and completely specified The verification results are reported in the work area A method that does not pass verification can be saved but cannot be run
145. les on the ligand surface so the bulk contribution to the relative response is smaller than on the reference surface As long as the refractive index of the samples is constant this excluded volume effect introduces a constant error in reference subtraction which may be ignored for practical purposes However if the refractive index of the samples varies the magnitude of the excluded volume effect will also vary Organic solvents like DMSO often give a high bulk response addition of 1 DMSO gives a bulk response of about 1200 RU so that small variations in the DMSO content lead to significant variations in the bulk response between samples Such variations are unavoidable in the preparation of diverse samples such as drug candidates for screening applications The solvent correction procedure corrects for the variations arising from the excluded volume effect in these cases A more detailed description of solvent correction background and procedures may be obtained from Biacore 6 7 2 When solvent correction should be used It is important to bear in mind that solvent correction is only relevant when e the expected analyte responses are low of the order of 50 RU or less e the ligand is a macromolecule immobilized at a high density typically 5 000 RU or more lower ligand densities lead to excluded volume effects that are too small to merit correction e the bulk response is subject to variations between samples of at least t
146. ligand immobilization concentration determination or measurement of kinetic constants Each wizard consists of an ordered series of dialog boxes ensuring that the essential features of the application setup are correctly defined Metbods provide greater flexibility and conversely less guidance in setting up applications allowing customized applications that are not covered by wizards Methods are defined in a graphical interface called Method Builder which is designed to provide full flexibility in method definition while retaining a simple interface for running assays based on established methods Application wizard templates may be opened in Method Builder to provide a starting point for further refinement of application setup Predefined methods are also provided by Biacore as help in defining methods for selected purposes Each of these modes of operation is described in more detail in the following chapters Biacore T100 Software Handbook 5 version AB Control Software general features 2 2 Userinterface The main screen in the control software is divided into 5 main areas with a separate event log window Menu and toolbar anti beta2micro Event lo E ESS 9 E Fs E O90 Few to levi 00 u 60000 4 Sensorgram window i m H 45000 40000 H f ady l 1 17600 Temperate 250 C 36000 V o
147. ll modify the injection procedure for better handling of viscous solutions The maximum injected volume is limited to 100 pl for viscous solutions Biacore T100 Software Handbook Version AB Application wizards 4 2 4 System preparations This dialog box specifies how the system will be prepared before the first cycle System Preparations xj IV Prime before run Normalize detector M Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Help lt Back Next gt Close Prime before run This option flushes the flow system with running buffer to make sure that all buffer is fresh You should generally prime the system before each run to ensure fresh buffer throughout the flow system Normalize This option adjusts the detector response to compensate for small variations in reflectance characteristics between individual sensor chips For best results you should normalize the detector whenever the chip is changed You do not need to run normalization if the same chip remains docked between runs Normalization injects BIAnormalizing solution 70 glycerol over the surface if your ligand does not withstand exposure to this solution normalize the detector before you run ligand immobilization Analysis temperature This is the temperature at the flow cell If the specified value differs from the current temperature the system will wait at the beginning of
148. lt Back Next gt Close Choose the flow path for the pH scouting Immobilization pH scouting is restricted to a single flow cell within a run The sensor surface in the flow cell should be unmodified Enter the buffers and pH values to be used for scouting The default list covers sodium acetate buffers in the pH range 4 to 5 5 available as ready to use solutions from Biacore Buffers will be tested in the order listed Biacore T100 Software Handbook Version AB Application wizards Note The buffers listed here are buffers in which the ligand should be prepared They are not used as running buffers you should use the same running buffer for pH scouting as you intend to use during immobilization 2 Injection parameters T Immobilization pH Scouting Injection Parai Pre xj Ligand Solution TestLigand Contact time 120 s Flow rate fio ul min Surface regeneration This surface wash will be run once at the end of each cycle Solution 50mM NaOH Help lt Back Next gt Close Enter the name of the ligand to be tested and the contact time and flow rate recommended contact time is 120 seconds you may need to use a longer contact time if preconcentration of ligand on the sensor surface proves to be slow The surface is washed with a regeneration injection at the end of each cycle to remove any ligand that might remain on the surface The recommended solutio
149. ly Samples and reagents are placed by category and categories are kept contiguous as far as possible Automatic Positioning xi Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Color Orientation Rack E cyan Bottom left Sample Small iS EX LightGreen E z Bottom left x Sample Small E DD zE Bottom left Sample Small E ghtGreen Bottom left Sample Small BH DarkCyan z Bottomleft z Sample Small E LightGreen Bottom left Sample Small Bottom left Sample Small Bottom left Sample Small Bottom left z Reagent Large Vial Size First Sort By Content Ascending Content Ascending Content Ascending Content Ascending Content Ascending Content Ascending Content Ascending Content Ascending Content Ascending enc cn Kc CC Crimson E LightGreen B crown NE Region This column lists the sample and reagent categories for the run Color This option controls the display color for the category Orientation This column determines whether samples are arranged by column vertically in the rack and plate diagram or row horizontally in the diagram Biacore T100 Software Handbook 29 Application wizards 30 Anchor This column determines the position for the first sample in the category Rack Th
150. m the Concentration Unit list The keyword values will be left unchanged but they will be interpreted for evaluation purposes in terms of the new unit For example a concentration entered as 10 pM will be evaluated as 10 mM if you change the concentration unit to mM If the evaluation session includes data from multiple files a table of concentration units for the different files is displayed Make sure that the unit is the same for all files if data are to be evaluated together Note The concentration unit affects only predefined concentration keywords Numerical user defined keywords are simply numbers and will not be re interpreted when you change the concentration unit even if they are intended to hold concentration information Biacore T100 Software Handbook Version AB Evaluation software general features Click Add Keyword to create a new keyword in the table You can choose between predefined keywords and user defined keywords see Section 5 6 2 If there are multiple Sample or General commands in the method from which the data is obtained specify the command to which the new keyword should apply Add New Keyword x M Keyword Type Pre defined User defined Apply to injection Sample 1 v Keyword name Diuton Value Type Test Numeric Ok Cancel Help Enter the required keyword values in the empty column that is created for the new keyword To rename o
151. ment Choose to set the zero response point to either a report point or an injection event If this setting is Off the actual response values will be shown If you check Enable Second Y Adjustment you can select a report point or injection event where the response value will be set to 100 Each sensorgram will then be normalized separately to the first and second adjustment point so that all sensorgrams will have values of 0 and 100 at these points regardless of the original response levels This can help in comparing the shapes of sensorgrams independently of their response levels or in adjusting response levels that are dependent on others e g adjusting analyte response for varying capture levels by adjusting the baseline to 0 and the capture level to 100 Biacore T100 Software Handbook Version AB Data presentation tools Blank subtraction Check Enable Blank Subtraction and choose a curve to be used as the blank to subtract one sensorgram from all others in the display Use this feature to eliminate systematic disturbances in sensorgrams that are not removed by reference subtraction Blank subtraction only affects the current sensorgram window other evaluation items are not affected Note Subtracting a blank sensorgram is not the same as using reference subtracted data Reference subtraction gives the difference between active and reference values for each cycle separately whereas blank subtraction subtracts one curve from all
152. ments 59 kinetics affinity application wizard 54 calculated values 136 137 evaluation 129 requirements for evaluation 86 129 subsets 137 kinetics affinity report 136 137 L labels in plot windows 97 legend 98 ligand 155 ligand immobilization 34 limit guides 142 linear fit 117 125 linked reactions 60 control results 62 local parameters 137 140 low molecular weight compounds 89 low sample consumption 77 M manual run 5 15 initial settings 16 markers 113 mass spectrometry 1 75 mass transfer 59 142 155 control results 61 mass transfer coefficient 144 mass transfer constant 143 144 Method builder importing wizard templates 63 Method Builder 5 63 commands 72 method examples 87 method overview 65 method structure 64 method variables 77 methods assay 5 folder 14 63 microplate 28 microplate in manual run 16 Microsoft Excel 171 mix and inject 38 mix function in concentration analysis 50 mixing sample before injection 77 Biacore T100 Software Handbook Version AB Error Reference source not found modification factor 142 molar concentrations 56 molecular weight of analytes 56 Multi detection 66 multi component complex 47 multiple ligand densities 131 137 evaluation 138 multiple result files 96 multiple Rmax 137 N nested assay steps 67 new cycle in manual run 19 normalize 27 normalizing sensorgram display 112 opening files for evaluation 96
153. mple Sample analysis Buffer A Sample Sample analysis Buffer A Sample Sample analysis Buffer A Sample Sample analysis Controls buffer A Control Sample Sample analysis Controls buffer A Control Sample Sample analysis Buffer A Sample Sample analysis Buffer A Sample Sample analysis Buffer A Sample Sample analysis i cme Enter the number of cycles to be run in each assay step and then click Simulate Cycle Run List to display the cycle sequence for the run as currently defined 5 5 1 Base settings Name This is the name of the assay step Each step in a method must have a unique name New steps are by default named Assay Step n where n is a serial number change the name to something that describes the context or intent of the step to make the method easier to follow Purpose Assay steps are assigned a purpose used to identify cycles in the evaluation software The choice of purpose can help to document the method structure and also determines the way the data is treated in evaluation Choose the purpose from the list An assay step may have one of the following purposes Purpose Usage and restrictions Calibration Used for calibration curves in concentration assays and affinity in solution This assay step should be connected to the same cycle type as the Sample step so that the calibration and sample analyses are performed in the same way Set Calibration to recurring within Sample to repeat the calibration at interval
154. mport to import the sample data from an external file Import of sample information must be enabled in Tools Preferences to use this function See Appendix A for details of import functions and file formats 7 System preparations Check the System preparations options as required see Section 4 2 4 8 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 9 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Biacore T100 Software Handbook 53 Application wizards 54 Evaluation Chapter 8 describes how to evaluate concentration assays 4 5 3 Kinetics Affinity The Kinetics Affinity wizard guides you through the setup of experiments to determine kinetic constants or affinity constants for an interaction Wizards for control experiments relevant to kinetic analysis are described in Section 4 6 1 Injection sequence Te Kinetics Affinity Injection Sequence xj Capture SAMPLE Sample REGENERATION 1 IM Regeneration E Help lt Back Nest gt Close Choose the injection sequence for the assay see Section 4 2 1 The Kinetics wizard supports capture but not enhancement injections The same injection sequence will be used for all selected assay steps 2 Assay setup Specify the flow p
155. n Chapter 3 of the Biacore T100 Instrument Handbook Set Temperature sets the sample compartment and analysis temperature More details are given in Chapter 3 of the Biacore T100 Instrument Handbook Preferences controls aspects of file storage and data import see Section 2 4 Biacore T100 Software Handbook Version AB Scale Copy Graph Export Curves Gridlines Control Software general features More Tools provides access to maintenance test and service tools Details are given in Appendix B of the Biacore T100 Instrument Handbook 2 3 7 Right click menus Right clicking with the mouse in some windows opens context menus specific for the window Sensorgram window Scale opens the same dialog as the View Scale option Section 2 3 4 Copy Graph copies the sensorgram window exactly as displayed to the Windows clipboard Use this option to insert a copy of the sensorgram window into other programs such as presentation software Export Curves exports data for the currently displayed curves to a text file Entire curves are exported regardless of the scale of the display The exported data includes report points and event marker times if these are displayed in the sensorgram window See Appendix A for more details of the export format Gridlines toggles display of gridlines in the sensorgram window Report point table The right click menu options for the report point table correspond to the Edit Report Poin
156. n for this procedure is 5 0 mM NaOH 3 System preparations See Section 4 2 4 Run immobilization pH scouting at the same temperature as you intend to run the immobilization electrostatic preconcentration is however usually fairly insensitive to temperature 4 Rack positions See Section 4 2 5 Immobilization pH scouting requires one position for ligand solution at each pH tested and one for the surface wash solution Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 5 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the start of a run when the wizard template is used Biacore T100 Software Handbook 33 version AB Application wizards 34 pH scouting results When the wizard run is completed the results are opened automatically in the Evaluation Software with an overlay plot of the sensorgrams adjusted to the start of the sample injection ixi 4 Curve Name Fe 1 z assay Step Purpose Sample e gt Zi Cycle Overlay Y v Tons gt RU Adjusted sensorgram EI Zoom Lock 5000 4 5000 4000 3000 4 A 10 mM Acetate 4 baseline 10 mM Acetate 5 2000 4 10 mM Acetate 4 5 10 mM Acetate 5 5 Response 0 1000 4 N 1000 4 2000 t t t t 1 10 40 90 140 190 240 Time 0
157. nalyte to the surface under the conditions of non turbulent laminar flow that prevail in the Biacore flow cell is characterized by the mass transfer coefficient k units m s1 pe vos BY z where D is the diffusion coefficient of the analyte f is the volume flow rate of solution through the flow cell h w l are the flow cell dimensions height width length One form used in fitting models in Biacore T100 is referred to as the mass transfer constant k units RU M 1 m s obtained by adjusting the mass transfer coefficient approximately for the molecular weight of the analyte and for the conversion of surface concentration to RU k 2k x MW x10 A further modification of this expression gives the flow rate independent component of the mass transfer constant units RU M s23m 3 referred to as tc in the models k tc XN 144 Biacore T100 Software Handbook Version AB 9 3 1 Kinetics 1 1 binding Kinetics and affinity analysis This is the simplest model for kinetic evaluation and is recommended as default unless there is good experimental reason to choose a different model The model describes a 1 1 interaction at the surface A B AB Model parameters are Obtained from surface RU ka Association rate constant M s Fitted kd Dissociation rate constant s Fitted Rmax Analyte binding capacity of the Fitted Conc Analyte concentration M Provided as input t
158. ndbook Version AB Error Reference source not found weight based concentrations 56 wizard templates 21 wizards importing into Method Builder 63 X XML 171 XML format 173 Z zero concentration samples 55 129 130 zoom 10 96 zoom lock 43 96 Biacore T100 Software Handbook 187 version AB Index 188 Biacore T100 Software Handbook Version AB BIACORE wwwW biacore com bR 1006 48
159. nding capacity of the surface in RU At molecular weight This parameters is only used in heterogeneous analyte models Check the box and enter the molecular weight parameter for the analyte to which the binding capacity parameter refers Binding capacity for the other analyte will be calculated using the molecular weight values Biacore T100 Software Handbook version AB 155 Kinetics and affinity analysis 156 Complex The complex is formed on the surface and generates response and has the properties listed below Generates Uncheck this box for complexes that form in response solution and that do not contribute to the response Molecular weight check this box and specify a parameter for complexes that form in solution and then bind to the surface Do not check this box if Generates response is also checked In the Bulk and Drift panel enter details for bulk refractive index contribution Normally there will be one bulk refractive index term applicable from the start to the end of the injection A second term can be used if necessary for example enter a second refractive index term with the injection end in the From box and the To box left blank to accommodate a permanent shift in baseline as a result of the sample injection Bulk And Drift Response RU From s To s IV Refractive index 1 RI t n S fort IV Refractive index 2 RI2 for Drift fin
160. ner of the sensorgram window to lock the scale to the current settings Adjust Scale sets the scale to the full data range This will not affect the Auto scale setting in the Scale dialog Adjust Scale overrides but does not turn off the Lock scale setting To scale the sensorgram display interactively drag with the cursor over the area to be scaled Double clicking in the display or choosing View Unzoom restores the previous zoom setting Biacore T100 Software Handbook Version AB Run Manual Run Wizard Method Control Software general features Reference line toggles display of a movable vertical line in the sensorgram window together with a separate small window that shows the response and time coordinates at the reference line for the current curve Use the Curve selector in the toolbar see Section 2 3 1 to set the current curve Drag the reference line to move it When the reference line is displayed choosing Baseline sets a baseline at the current reference line position and the coordinates window shows the response relative to that baseline The options Show Only Current Curve Show Curves of Same Type and Show All Curves control which curves are displayed in the sensorgram window Curve types distinguish between unsubtracted and reference subtracted curves Choose the Event Log option or click on the Event Log button at the right of the toolbar to display the event lo
161. ning analyte Only Sample commands are recognized in the evaluation software for kinetics affinity and concentration evaluation The injected solution contact time dissociation time and flow rate can be set as variables Evaluation variables can also be defined for the Sample command see Section 5 6 2 The Sample command may be optimized for high performance or low sample consumption High performance is achieved by using extra segments of air and sample during aspiration to separate the injected solution from running buffer thereby minimizing dispersion of sample at the beginning and end of the injection at the expense of additional 25 pl sample consumption The low sample consumption option uses fewer segments but still achieves a performance that is adequate for all applications except analysis of rapid kinetics The Sample command supports a Mix function for mixing sample with a defined solution in the autosampler before injection Check the Mix option and enter a mix solution and mixing fraction to use this function Entering a fraction of e g 20 will mix one part of mixing solution with four parts of sample The sample and mixing solution are taken from respective positions in the autosampler and mixed in a third position The option Stabilization period after mix allows you to specify a wait period between the mixing operation and injection of the mixed solution Solvent correction command This command injects a 30 second pulse o
162. njection see Section 7 1 3 Biacore T100 Software Handbook Version AB 5 Methods Methods in Biacore T100 offer flexibility in instrument control providing support for applications that cannot conveniently be handled with wizards Methods are constructed with the Method Builder tool as described in this chapter Templates from application wizards can be opened in Method Builder Section 5 1 to provide a starting point for development of customized applications Method examples are provided with the software installation 5 1 Opening methods To open an existing method or create a new method choose File Open New Method lolx Look in ja Methods And Templates amp c woo Teile E BufferChange Method Builder 17 Feb 05 5 KineticsDiffBuffers050209 Method Builder 09 Feb 05 Help Browse Show importable wizard templates New Cancel A Select a method and click Open to open the method or click New to create a new method Predefined methods for common applications are provided in the folder Biacore Methods If you make changes to a predefined method you must save your changed method under a new name The top level folder for methods is defined under Tools Preferences see Section 2 4 You can navigate between subfolders under the top level in the dialog box but files outside the top level folder are not listed in the dialog box Click Browse to navigate freely in the computer file structure and open m
163. nning of the run T Concentration Analysis Setup x Detection Flow path 12 hd Startup IV Run startup cycles Solution Dummy Number of cycles 3 Help Flow path Select the flow path for the analysis The setting will apply throughout the whole wizard run lt Back Next gt Close The flow paths available vary between the different wizards The detection is automatically set to the same settings as the flow path so that sensorgrams are recorded only from the flow cells used When reference subtraction is used together with ligand capture Section 4 2 1 the captured ligand passes over the active surface but Biacore T100 Software Handbook Version AB Application wizards not the reference surface for example with Flow path set to 2 1 the ligand is injected in flow cell 2 but not flow cell 1 If the Flow path setting does not use reference subtraction ligand is injected in all flow cells included in the flow path Start up cycles Start up cycles are identical to analysis cycles except that the sample is replaced by a dummy sample The response from a newly prepared or newly docked sensor chip often shows some instability during the first few cycles and start up cycles allow the response to stabilize before the first analysis cycle is performed Three start up cycles are generally recommended for most assay purposes to ensure a stable response in the analysis St
164. ntration C The equation includes a term for the bulk refractive index contribution RI which is assumed to be the same for all samples This term simply serves as an offset on the Reg axis CR id E max 4 RT Kp C Model parameters and reported results are Obtained from KD Equilibrium dissociation constant M Fitted Rmax Analyte binding capacity of the Fitted surface RU RI Bulk refractive index contribution in Fitted the sample 152 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis Note Reported Kp values that are higher than half the highest analyte concentration used should be treated with caution If the response against concentration plot does not flatten out sufficiently because the concentrations are not high enough in relation to the Kp value the reported value may be unreliable The reported Kp value is marked as a vertical line on the fitting plot see Section 9 1 2 9 4 Creating and editing models To create your own models for kinetics of affinity evaluation choose Tools Models from the main menu and select the type of model you want to work with You can use existing models as templates Choose an existing model from the list and click New answer Yes in the following dialog to create a new model based on the chosen template or No to create a blank model For kinetic models you can define a new model either as a reaction scheme describing the
165. o not affect the data set that will be evaluated If you have multiple ligand densities represented in the result set click Multiple Rmax to assign curves to the different sets see Section 9 1 3 Click Adjust Injection Start if the sample injections do not start at precisely zero time The injection start is set automatically from the event marker but may need slight adjustment for best fitting results The adjustment compensates for small systematic discrepancies in the interval between the start of the injection as recorded in the event log and the time that the same actually reaches the sensor surface The difference is most apparent at low flow rates The discrepancy has very little effect on the rate constants obtained from the fitting except for very fast reactions and adjusting the injection start is seldom necessary Biacore T100 Software Handbook 131 version AB Kinetics and affinity analysis 132 EB Modify Injection Start x I Zoom lock Drag the vertical reference line to adjust the injection start point You can adjust the start by 10 s from zero The same adjustment is applied to all curves in the data set whether they are currently included for evaluation or not Click Next gt when you have selected the curves to be evaluated The second dialog shows the blank subtracted curve set and allows you to delete selected regions from all or selected curves for example to eliminate spi
166. on 75 detection 66 in wizards 24 diffusion coefficient 144 diluting ligand for immobilization 36 diluting ligands for immobilization 38 dilution factor 53 79 127 dimethyl sulfoxide 103 dissociation time 55 DMSO 103 drift 156 Dual detection 66 dual inject 75 Biacore T100 Software Handbook 179 version AB Index 180 E Edit menu control software 9 editing keywords 101 editing report points in control software 9 in evaluation software 100 eject rack tray manual run 18 ending a manual run 19 20 enhancement 24 74 plot 99 enthalpy change 161 entropy change 161 epitope mapping 47 equation models 158 evaluating concentration analysis 123 evaluation of kinetics and affinity 129 evaluation requirements 85 evaluation software general features 95 screen regions 96 evaluation variables 78 101 event markers 113 Excel 171 excluded volume effect 104 excluding data from concentration analysis 125 126 127 excluding data from evaluation 97 excluding data from kinetics affinity evaluation 130 excluding data from plots 116 expected concentration 126 export curves 13 98 export to Excel 171 export to XML 171 exporting data 171 exporting plots 98 exporting rack positions 31 172 exporting the report point table 172 extra wash after injection 72 extrapolating solvent correction curves 107 Eyring equation 162 F File menu control software 7 file name extensions 14 fil
167. on curve does not fit the experimental points closely Scatter in the correction points indicates that the measurements are not reliable and applying correction derived from such curves can distort the measured responses unnecessarily Biacore T100 Software Handbook Version AB Evaluation software general features The range of report point values that are candidates for solvent correction in the assay data is indicated by vertical red lines in the window If report points lie outside the correction range these values cannot be properly corrected Some small extrapolation of the correction plots may be permissible Use the Extrapolate button to extend the correction range The shape of the solvent correction plots is however not fully predictable and extrapolation over more than a short distance at most 10 of the range of the reference values is dangerous Click OK to apply the solvent correction Correction will be applied to the sample and carry over injection phase s of all sensorgrams Any data points that lie outside the correction range will be discarded and the corresponding sensorgram will contain gaps corresponding to the invalid data Biacore T100 Software Handbook 107 version AB Evaluation software general features 108 Biacore T100 Software Handbook Version AB Data presentation tools 7 Data presentation tools This chapter describes the tools available for presentation and examination of the data in a re
168. opened automatically in the Evaluation Software with overlay plot of adjusted sensorgrams according to the control experiment Mass transfer The sensorgrams are adjusted to zero response and time at the baseline report point Compare the observed binding rates at the different flow rates Biacore T100 Software Handbook 61 version AB Application wizards 62 EB all sensorgrams Curve Name Fo 4 3 v 4l Assay Step Purpose Undefined z 4l Cycle lt Overly gt m Tools RU Adjusted sensorgram Zoom Lock lb 400 4 300 4 200 4 Response 0 baseline 100 4 100 t t t t t t t 1 150 100 50 o 50 100 150 200 250 Time 0 baseline Linked reactions The sensorgrams are adjusted to zero response at the baseline report point and to zero time at the end of the sample injection Compare the observed dissociation rates at the different contact times LI ox cuve Name Fo 21 n Assay Step Purpose Undefined z w foce oves m Tek RU Adjusted sensorgram ET Zoom Lock 1600 4 1400 4 1200 4 1000 4 800 4 Response 0 baseline 4004 200 4 200 t t t t t t t t 1 1000 800 600 400 200 o 200 400 600 800 Time 0 Sample 1 stop Note If the binding does not reach a steady state in all sensorgrams as in the example above it may be easier to compare the dissociation rates if you use a second y adjustment point at the end of the sample i
169. ounds that may affect the response in subsequent injections The Biacore method is designed for screening of low molecular weight compounds and includes a solvent correction step as described in Section 5 11 3 Low molecular weight compounds frequently dissociate readily from their targets and the example method does not include regeneration If you require a regeneration step it is advisable to include regeneration after both the sample and the carry over injections i LMW screen Main EXTERNER TEENS SONO Cycle type description General Settings Cycle types currently in method New This cycle is used in startup sample and control sample steps EE Contains injection of sample and carry over control running buffer solvent correction REUS sd LMW screen lt a gt m Sample amp Ic Verification 2 Insert the required commands LL 3 Configure settings for each command Beinen Captum E Settings for Cany over control 1 eem The Carry over control injection wil be run for 30 seconds with a flow rate 40 ul min Remove From Cycle Move Up Move Down Sample 1 Carty over control 5 11 5 Capture on Sensor Chip NTA Capture of poly histidine tagged ligands on Sensor Chip NTA relies on chelation of Ni ions on the surface and regeneration is generally performed by stripping the Ni2 with EDTA Applications that use this approach require two capture injections in each cycle one for Ni and one for th
170. p M Wizard Results C cres Include event log for cycles ted Sensorgrams will be printed as follows None No sensorgrams will be printed Current cycle The current cycle will be printed with the View Show setting and scale as shown on the screen Range and Multiple cycles will be printed For Range enter a All cycles range or cycle numbers separated by commas e g 4 16 19 22 All curves will be included in each cycle regardless of the View Show setting Sensorgrams will be printed at full scale unless the Lock Scale box is checked in the sensorgram window in which case the current scaling will be applied to all cycles with this setting some sensorgrams may appear to be empty Note In order to maintain report layout the print orientation is fixed regardless of the printer settings in Windows Properties shows detailed properties of the currently opened run including the properties of the sensor chip used in the run When you close the software with Exit while the instrument is still switched on you may choose to shut down the instrument for a shorter or longer period if required 8 Biacore T100 Software Handbook Version AB Control Software general features Exit Software x What do you want to do with the instrument Leave instrument in standby mode C Shut down instrument For a long term period C Exitthe software This leaves the instrument in standby mode
171. p cycle will be run with buffer for all pulses Help lt Back Next gt Close Regeneration scouting always starts with one startup cycle with the same injection sequence as the scouting cycles but with injection of buffer for all injections Note that this differs from the startup cycle construction in the other application wizards Biacore T100 Software Handbook Version AB Application wizards 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 The same sample will be used for all cycles 4 Experimental parameters T Regeneration Scouting Experimental Parameters xj Regeneration parameters Flow rate 20 pl min Stabilization period o s High viscosity solution Experimental design Number of conditions 4 Lock Solutions v Contact times Number of cycles for each condition s Settings Condition Regeneration solution Contact time s 4 N lt Back Next gt Close This dialog box determines the design of your regeneration scouting Set the number of conditions to test and the number of cycles for each condition and specify the conditions in the Settings frame The default number of cycles for each condition is five You may use fewer cycles to shorten the total run time for exploratory work but five cycles are recommended for fine tuning conditions in order
172. parations Starting a manual run Controlling a manual run Ending a manual run 4 Application wizards 4 1 4 1 1 4 1 2 4 2 4 2 1 4 2 2 4 2 3 4 2 4 4 2 5 4 2 6 Biacore T100 Software Handbook version AB Wizard templates Creating and editing wizard templates Running wizards Common wizard components Injection sequence Assay setup Injection parameters System preparations Rack positions Prepare Run protocol Contents o2 15 15 15 16 17 20 Contents ii 4 3 Surface preparation wizards 4 3 1 Immobilization pH scouting 4 3 20 Immobilization 4 4 Assay development wizards 4 4 1 Regeneration scouting 4 4 0 Surface performance 4 4 3 Buffer scouting 4 5 Assay wizards 4 5 1 Binding analysis 4 5 2 Concentration analysis 4 5 3 Kinetics Affinity 4 5 4 Thermodynamics 4 6 Control experiment wizards 4 6 1 Mass transfer control 4 6 2 Linked reactions control 4 6 3 Evaluation of control experiments Methods 5 1 Opening methods 5 2 Method structure 5 3 Method overview 5 4 General settings 5 5 Assay steps 5 5 1 Base settings 5 5 2 Number of replicates 5 5 3 Recurrence 5 5 44 Assay step preparations 5 6 Cycle types 5 6 1 Commands 5 6 2 Variables 5 6 3 Report points 5 7 Sample and Assay Setup 5 8 Verification 5 9 Setup Run 5 9 1 Detection 5 9 2 Variables 5 9 3 Cycle run list 5 9 4 System preparations 5 9 5 Rack positions 5 9 6 Prepare Run P
173. period fao s Help lt Back Next gt Close This feature enables inhibition assay formats where samples are mixed with a constant proportion of a detecting molecule solution The value specified for Fraction refers to the proportion of the fixed component in the final mixture for example a value of 30 will mix 7 parts of sample with 3 parts of the specified solution The volumes of sample and mixing solution used are determined automatically so that the final volume of mixed solution is sufficient for the injection Notes The mixing function is not supported in 384 well microplates The wells on these plates are too small for reliable mixing with the autosampler needle Mixing in the autosampler is very reproducible but high accuracy cannot be guaranteed If your application requires accurate mixing proportions mix the samples outside the autosampler Biacore T100 Software Handbook Version AB Application wizards 4 Calibration curve T Concentration Analysis Calibration Curve x m Calibration Curve Analyte name Standard v Repeat calibration every fio sample cycle m Calibration points Concentration El Help lt Back Nest Close Specify the details of the calibration curve for the concentration measurement Check Repeat calibration and enter a number of sample cycles to repeat the calibration curve at intervals during the assay The calibr
174. pl ligand solution at a flow rate of 5 pl min giving a contact time of 2 minutes This injection is included in predefined methods for CM series sensor chips but is optional in customized methods see below If Aim for immobilized level is chosen together with a custom method that does not include a preconcentration injection the immobilization procedure will activate the surface and then inject short pulses of ligand until either the target level or the maximum total ligand volume of 150 pl is reached This option can be used to conserve valuable ligand without losing the benefits of aiming for a target immobilization level This option is particularly useful for sensor chips where preconcentration cannot be performed e g Sensor Chip SA Biacore T100 Software Handbook Version AB Application wizards Custom methods Click Custom Methods to define customized immobilization methods ixi Methods T P PRE CONC A MIXINJECT JC NHS 50 50 20 5 nem 2 A WASH Ethanolamine LD LIGANDINJECT Specified in Immobilization Setup Mix amp Inject 5 4 mcr Ethanolamine 420 10 vum Delete D oee Move Up ED Move Down OK Cancel yy Click New to create a new blank method Select an existing method and click Copy or Delete to make a copy of the method or delete it respectively You cannot delete the predefined methods marked with a 33 icon For a new method enter a name in the Method name field Con
175. plot This cycle is shown as cycle number 2 cycle 1 is the startup cycle that is not shown in the plot Select which conditions to display in the Conditions box Use Shift click to make multiple adjacent selections Ctrl click to make multiple non adjacent selections The scale of the display will be adjusted according to the number of cycles displayed Select which curves to display in the Sensorgram box The Sensorgrams tab shows the sensorgrams for regeneration scouting Select the conditions and cycles to display in the respective boxes Check Zoom lock to keep the scale fixed when the choice of sensorgrams is changed Result files from regeneration scouting can also be opened in the Evaluation Software if you want to prepare other sensorgram displays or plots see Chapter 7 4 4 2 Surface performance The surface performance wizard allows you to test the robustness of your surface by performing repetitions of the same analysis cycle The number of repetitions is in practice limited by the capacity of the sample plate the software allows up to 400 cycles The cycle requires one sample injection and can also include capture and enhancement steps and one or two regeneration steps Use this wizard for example when you want to confirm that the regeneration conditions that you identified in regeneration scouting hold good for an extended number of cycles 1 Injection sequence Choose the injection sequence for the surface performanc
176. porting data 172 A 2 1 Control Software Sample table import The Control Software supports data import to sample tables in assay wizard templates and in the Setup Run step of methods In order to use the import function the option must be activated in Tools Preferences and an import program must be specifed x Folders Import IV Enable sample information import Program C Program Files T 100S amplelmport exe E v Enable rack positions import Program C Program Files T 100Position mport exe Py Biacore T100 Software Handbook Version AB Affinity in solution When the import function is requested from a dialog box the contents of the table are first exported in Extended Markup Language XML format to a temporary file that is submitted to the specified import program The import program may append new sample data to the file or overwrite the file contents with new data as required The modified file is then imported back into the sample table and the temporary file is deleted Development or choice of a suitable import program is the responsibility of the user To document the detailed XML format of the import file specify an XML compatible text editor as the import program and save a copy of the import file from a suitable table lt xml versionz 1 0 encoding is ETT lt DOCTYPE MethodBuilderImport Source for full doctype MethodBuilderImport importFileVersion 1 0 gt lt AssaySteps gt
177. r delete a keyword click the appropriate button then select the keyword in the dialog box You cannot remove system generated keywords such as file number or cycle number or keywords derived from method variables such as sample name 6 7 Solvent correction 6 7 1 Background Solvent correction adjusts reference subtracted responses for small artefacts that can be introduced by variations in the bulk refractive index between samples The correction is only relevant when variations in the bulk refractive index are of the same order of magnitude as the response this situation arises commonly in work with small organic analytes that give intrinsically low response values and that often require solvents such as dimethyl sulfoxide DMSO to maintain solubility The need for solvent correction arises because subtraction of the reference response does not exactly eliminate the contribution of the bulk solution to the measured response Bulk solution is excluded from the volume occupied by ligand on the active surface so that the bulk contribution to the response on the active surface is slightly smaller than that on the reference surface see illustration Biacore T100 Software Handbook 103 version AB Evaluation software general features 104 Response on reference surface Response on ligand surface X oe a n n Reference surface Ligand surface Bulk solution is excluded from the volume occupied by ligand molecu
178. range e g 10 40 See Section 10 1 for further details 58 Biacore T100 Software Handbook Version AB Application wizards Specify the sample compartment temperature or check Vary with analysis temperature to change the sample compartment temperature automatically when the analysis temperature is changed J Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 The sample injection for thermodynamics measurement has an additional setting for dissociation time This is the time for which dissociation will be monitored after the end of the injection without disturbances from flow system washing procedures 4 Sample parameters Enter the details of the samples This dialog box is equivalent to the concentration series dialog for kinetics determination see Section 4 5 3 step 4 Separate rack positions will be created for each sample at each temperature 5 System preparations Check the System preparations options as required see Section 4 2 4 The settings for analysis temperature and sample compartment temperature cannot be changed here the values for the first temperature as specified in step 2 are shown 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displ
179. riod If you choose a command sequence for either the True or False outcome click on the appropriate branch of the command Then or Else respectively and insert the commands you wish to be executed If you leave the branch empty the cycle will simply continue with the next command following the If then construction You can use the Move up and Move down buttons to rearrange the order of commands within a branch but you cannot move commands outside the branch in which they are placed If you have chosen a command sequence for an action and have entered commands you must delete the commands before you can change to a different action Enhancement command This command is intended for injection of a secondary enhancement reagent following the sample injection Enhancement reagents are most commonly used to confirm the identity of the bound analyte but may also be used to amplify the analyte response The injected solution contact time and flow rate can be set as variables Biacore T100 Software Handbook Version AB Methods General command This command is a general purpose injection that supports the same functions as the Sample command and also includes a Dual Inject option under Type This option injects two solutions in direct succession with no intervening automatic wash routines A dissociation time may be set for the second injection but not for the first General commands are not recognized as analyte injections for ev
180. rotocol 5 9 7 Starting the run 5 10 Requirements for assay specific evaluation 5 10 1 Concentration 5 10 2 Kinetics Affinity 5 10 3 Thermodynamics 5 10 4 Affinity in solution 5 10 5 Other requirements 32 32 34 40 40 43 45 47 47 49 54 57 59 60 61 61 63 63 64 65 66 67 68 69 70 71 71 72 77 79 81 82 82 82 82 83 84 85 85 85 85 85 86 86 86 87 Biacore T100 Software Handbook Version AB 5 11 5 11 1 5 11 2 5 11 3 5 11 4 5 11 5 5 11 6 Method examples Affinity in solution Heterogeneous analyte kinetics Low molecular weight compounds Screening Capture on Sensor Chip NTA Inject and Recover 6 Evaluation software general features 6 1 6 2 6 3 6 3 1 6 3 2 6 4 6 4 1 6 4 2 6 5 6 6 6 7 6 7 1 6 7 2 6 7 3 6 7 4 Evaluation software interface Opening files Common display functions Zooming the display Right click menus Predefined evaluation items Sensorgram Plots Custom report points Keywords Solvent correction Background When solvent correction should be used How solvent correction works Applying solvent correction 7T Data presentation tools 7 1 7 1 1 7 1 2 ed 7 1 4 7 2 7 2 1 7 2 2 7 2 3 7 2 4 7 3 7 3 1 Sensorgram windows Selecting sensorgrams for display Removing data Sensorgram adjustment Markers Plot windows Selector functions Table functions Fitting curves to points Ranking Report point table
181. rpose Sample Conc uM Reset All Fiters Bm sm i g E BELOW 1i Conditioning buffer 2 Conditioning buffer 3i Conditioning buffer i Add Keyword 4i SolventCorrection 5i Sample Furosemide2 Rename Keyword Bl Sample Furosemide 0 7 Sample Furosemide1 0 3125 Remove Keyword 8i Sample Furosemide2 0 3125 H Sample Furosemide1 10 10 Sample Furosemidel 0 11 i Sample Furosemide 12i Sample Furosemide1 0 1563 5 13 Sample Furosemide2 0 SCENES 1 H Sample Furosemide2 1 25 uM E 15 Sample Furosemide2 0 525 16 Sample Furosemide2 5 17 SolventCorrection 18i Sample Furosemide2 25 13 Sample Furosemide 1 25 20 Sample Furosemide 0 625 21 i Sample Furosemidel 5 22 Sample Furosemide1 25 23i Sample Furosemide2 0 To simplify management of the keyword table you can sort and filter the table display e Click on a column header to sort the table by the contents of that column e Click in the filter row directly below the column header and select a value to display only rows with that value for the chosen column Click Reset All Filters to restore all filters to the AII setting To change a keyword value simply enter the new value in the appropriate cell Values for some system generated keywords such as Assay Step Purpose are chosen from a predefined list of values the list is displayed when you place the cursor in such a cell To change the units for concentration keywords choose a new unit fro
182. rranged in pairs Fc1 2 and Fc3 4 with minimum dead volume between the flow cells in a pair to provide accurate reference subtraction e The sample compartment accommodates one microplate 96 or 384 well regular or deep well capacity and one reagent rack for reagent vials A combined sample and reagent rack can be used in place of the separate microplate and reagent rack e Material that binds to the sensor surface during sample injection can be recovered in a small volume of liquid for further analysis by e g mass spectrometry e The temperature in the sample compartment is controlled separately from the analysis temperature allowing samples to be kept at one temperature while analysis is performed at another Samples equilibrate to the analysis temperature during injection into the flow cell The analysis temperature can be varied during a run e The system includes a buffer selector valve allowing analysis to be performed in up to four different buffers in the same unattended run Biacore T100 Software Handbook 1 version AB Introduction 1 2 Support for use in regulated environments Support for use in regulated GxP environments is provided in an optional package that adds appropriate functionality to the Biacore T100 software Functions for GxP support are described in a separate Biacore T100 GxP Handbook Descriptions of software in the current Handbook apply to installations with and without the GxP package unless oth
183. rve parameters under the Tools button to toggle display of the fitting parameters below the plot panel Biacore T100 Software Handbook 117 version AB Data presentation tools 7 2 4 Ranking Choose Ranking under the Tools button to add ranking boundaries to the plot You can add one or two boundaries classifying the plot points as Low High or Low Medium High The boundary values are specified in RU New Ranking r Ranking Settings C None C One Ranking Boundary Two Ranking Boundaries M Ranking Boundaries First Value 5 Second Value 25 Help Finish Cancel Boundaries are shown as horizontal red lines in the plot labeled with the boundary value The classification of the points is recorded in the table BB Binding levels Sample nl x 4 Curve Name Fc 2 1 4l Assay Step Purpose Sample z E Cycle number Overlay kA v Tools w RU Binding levels Sample Zoom Leck Borm Y Value Ranking 4 40 4 25 2 208 Low 24 1 813 Low 23 1 840 Low 30 4 22 1 676 Low Value 25 21 1 562 Low A 20 0 543 Low 20 19 23 266 Low 18 21 120 Medium 17 16 327 Medium 16 11 581 Medium 15 0 128 Low 2 420 Low teste 13 29 195 High 12 22 827 Medium 404 11 15 474 Medium 10 7 872 Medium 9 5 420 Low 8 2 463 Low r 28 208 High 6 22 841 Medium 5 4 i 4 Value 5 Relative response stability eai o
184. s definitions see Section 5 6 1 Miscellaneous settings Enter names if desired for the buffers in bottles A to D Names entered here will be displayed in the Prepare Run Protocol Section 4 2 6 Different buffers may be chosen for different assay steps as described in Section 5 5 4 the default choice is bottle A Te BufferChange Main 1 Create assay steps by using the buttons below Startup Startup Sample analysis 3 times as entered 2 Select an assay step by p e x below or use buttons lo Buffer A Cycle Types remove or move the step Sample Sample analysis 1 time as entered Sample amp Save as New Step t Remove Step Move Up t Move Down Controls buffer A Control sample Sample analysis 1timeasentered Before every 10 cycles DMSO buffer A Solvent Correction Solvent correction 1 time as entered Before every 15 cycles Buffer B sample Sample analysis 1 time as entered Copy Step Simulate Cycle Run List t gj Controls buffer B Sample analysis 1 time as entered Before every 10 cycles Control Sample Controls buffer A Papot Control sample z Connect to Sample analysis E Recurrence I Repeat assay step within Buffer A po c occurences evenly T Run assay step once last C Eve C Distibute TV Run assay step once first cycle type Number of replicates Assay step prepara
185. s entered the right hand Concentration column displays the conversion from molar to weight based or vice versa Samples with the same sample name may not be given different molecular weights The samples may be analysed either in the order entered in the table or sorted in increasing concentration The order displayed in the sample table is not affected by the choice of run order If you enter samples with different names they will be handled as separate concentration series regardless of the order in which they are entered The samples will be run as separate concentration series even if the order is mixed in this dialog thus samples entered in the order A B A A B A B B will be run in the order A A A A B B B B The Run order setting applies within each concentration series Click Import to import the sample data from an external file Import of sample information must be enabled in Tools Preferences to use this function See Appendix A for details of import functions and file formats Click Control Samples to enter control samples for the run Ts Kinetics Affinity Control Samples 1 x M Control sample definition v Run control samples v Repeat control sample s every fi 0 sample cycles M Control samples Control sample id 1 high low CIN Help Cancel OK Biacore T100 Software Handbook Version AB Application wizards Specify the details of control samples and
186. s or by editing the keyword table Changing sample names in calibration cycles has no effect on the calibration curve 8 2 2 Control samples The Control Samples tab displays the measured concentration for control samples as a trend plot of response against cycle number Biacore T100 Software Handbook 125 version AB Concentration analysis 126 x Calibration Control Samples Samples Control sample table Control sample plot Comp to nginl oge samena aare a n eem 10 Control 1 33 3 608 0 33 52 100 7 1 17 Control 1 33 3 609 2 33 4 100 3 1 24 Controli 33 3 593 0 35 07 105 3 1 354 31 Control 333 6l41 32 92 9895 1 N T aj 44 Control 1 33 3 600 5 34 01 102 1 2 51 Control 1 33 3 608 7 33 2 99 71 2 Avg 33 69 2 288 101 16 zd 11 Control2 11 1 996 1 10 71 96 5 1 18 Control 2 11 1 992 4 10 84 97 66 1 25 25 Control2 at 984 3 11 13 100 2 1 32 Control2 11 1 989 1 10 96 98 7 1 45 Control2 111 989 2 11 35 102 3 2 52 Control2 11 986 8 11 43 103 2 a Avg 11 07 2 576 99 73 e t T 10 t 1 1 1 A E S aaa Saa 5 10 15 20 25 30 35 40 45 50 55 Cycle Li Control sample concentrations are calculated from the nearest preceding calibration curve or the nearest following if there is no preceding curve Numerical results are presented in the table at the left and plotted as calculated concentration again
187. s through the assay At least one calibration step is required for evaluation of concentration analysis Conditioning Used to condition the sensor surface at the start of an assay 68 Biacore T100 Software Handbook Version AB Methods Control sample Used for control samples This assay step should be connected to the same cycle type as the Sample step so that the control sample and sample analyses are performed in the same way Set Control sample to recurring within Sample to repeat the control sample analysis at intervals through the assay Sample Used for sample analysis in all applications At least one sample step is required for application specific evaluation Solvent correction Used for solvent correction cycles This step should be connected to a cycle type designed for solvent correction Set Solvent correction to recurring within Sample to repeat the solvent correction at intervals through the assay Startup Used to condition the flow system at the start of an assay This assay step will commonly be connected to the same cycle type as the Sample step Undefined Used for assay steps that do not fit the predefined purposes Assay steps with Undefined purpose will not be included in application specific evaluation Note For many simple methods the assay step name and purpose may often be the same e g Solvent Correction Sample Control Sample etc It is ho
188. sensorgram 3 The reference subtracted sensorgram is corrected by subtracting the solvent error This procedure is applied to every point during sample injection Reference Fc Solvent correction is applied only to response levels during sample injection since the correction adjusts for differences in the bulk refractive index of the samples Before and after sample injection when buffer is flowing over the surface the bulk response is constant and correction is meaningless 6 7 4 Applying solvent correction To apply solvent correction choose Add solvent correction from the Evaluation menu In order to apply solvent correction the run must include solvent correction cycles see Section 5 6 1 Biacore T100 Software Handbook 105 version AB Evaluation software general features 106 x RU Included Cycle Curve Chiz RU YO RU Zoom lock Mod fees 1 137 34 zm i 17 Fe 4 3 0 94 4 0 v 2q NN V NN NN Y NN 3 N x Y RN eS N a Y B 8 S N oS H N Be YN N v 3 x N N o 64 X N N LA v v r Extrapolation Range Report Point Range 7 t t t T t t t t t 1 800 600 400 200 0 200 400 600 800 1000 1200 Extrapolate v None V Show report point range Response Ref RU Help The left hand panel of this dialog lists the solvent correction curves in the run and the corresponding solvent correction curves are shown in
189. st cycle number on the right Select a row in the table to highlight the corresponding point on the plot The table lists the expected concentration as entered for the control samples the response and calculated concentration the calculated concentration as a percentage of expected and the calibration curve used to calculate the concentration Replicate control samples are summarized with average values and coefficient of variation CV for the response and calculated concentration Right click on a sample row in the table or a sample point on the curve and choose Exclude Cycle to exclude that sample from the sample evaluation Excluded cycles are shown as open symbols and are marked in the table with red strikethrough text Biacore T100 Software Handbook Version AB Concentration analysis 8 2 3 Samples The Samples tab displays the measured concentrations for samples E Concentration Analysis Create xi Calibration Control Samples Samples m Sample table m Sample plot Cycle Sample Id Dil Response Calc Conc cv Calib RU Biotin Fact RU ng ml 9o Curve 1400 4 12 501 2436 01 1 1001 1 10 54 1 13 501 2436 01 1 987 4 11 02 1 14 501243601 2 11737 10 97 1 4205 15 501 2436 01 2 1168 6 11 23 1 Avg 10 94 2 640 16 501 2436 02 1 606 8 33 64 1 1000 7 19 501243602 1 607 1 33 62 1 Avg 33 63 0 04622 y 1E 800 J 1 20 501 2437 01 1 808
190. struct the sequence of injections for the immobilization method using the buttons to the right of the main panel The ligand injection is created automatically and cannot be deleted solution and contact time for the ligand injection are specified in the main wizard dialog A method may only contain one ligand injection Other injections have the following functions Pre conc injects 10 pl of ligand solution at 5 pl min to estimate the rate of preconcentration This step is only performed if the option Aim for immobilized level is chosen when the immobilization method is used A method may only contain one Pre conc injection The Pre conc injection should always be placed before surface activation it will usually be first in the method although it may be preceded by a surface conditioning injection if required If you place the Pre conc injection after the surface activation it will be executed there and the ligand will be immobilized on the activated surface Biacore T100 Software Handbook 37 version AB Application wizards 38 After the Pre conc injection the surface is washed with a solution specified in the immobilization setup dialog to remove any ligand that may remain on the surface Do not use a Pre conc injection with Sensor Chip SA since biotinylated ligand will bind to the surface and cannot be removed Inject performs an injection of a specified solution with a specified contact time and flow rate Values are entere
191. sult set These tools comprise e Sensorgram display with facilities for aligning sensorgrams in overlay plots e Plot tools for displaying and ranking response values e A report point table for listing numerical values associated with report points 7 1 Sensorgram windows Sensorgram windows display the sensorgrams from one or more cycles in the result set To create sensorgram display window click Sensorgram in the toolbar or choose Add sensorgram from the Evaluation menu The window is created directly with an overlay plot of all cycles in the result set You can open any number of sensorgram windows with independent display settings The window is listed in the evaluation explorer as Sensorgram n where n is a serial number Right click on the sensorgram item in the evaluation explorer and choose Rename to change the item name ini x Bi Curve Name Fo 4 3 Leh Assay step Purpose lt Overlay gt m t amp fCycle lt Overlayy m Tools v RU Adjusted sensorgram I Zoom Lock Response 0 baseline 1 Biacore T100 Software Handbook 109 version AB Data presentation tools 4 w 110 Hold the cursor over a sensorgram to display a tool tip identifying that particular curve The sensorgram coloring can be changed if desired with the Tools Color by option The following sections describe display functions specific to sensorgram windows General display functions are described in Section 6 3 7
192. t for different curves and bulk refractive index contribution which may be expected to vary between curves e Global parameters have one single value that applies to the whole data set Typical global parameters are the rate constants for the interaction which should in principle have the same value for all curves in the data set e Constants have a fixed value that is not changed in the fitting procedure An example is the analyte concentration Constants may also be local separate values for each curve or global one value for the whole data set The local global status of parameters can be changed through the Parameters button in the fitting dialog step 4 in Section 9 1 2 above without making any changes to the model Evaluating kinetics or affinity with global rate constants gives a more robust value for the rate constants although the curves may fit the experimental data more closely if all parameters are fitted locally This is because local fitting allows variation between the constants obtained from different curves when the constants are fitted globally this variation appears in the closeness of fit rather than the reported values Rate constants are always global in predefined kinetic models In general kinetic constants should be fitted as global parameters and bulk refractive index contribution as a local parameter The analyte binding capacity of the surface Rmax is a global parameter by default in the predefined mod
193. t the analysis and sample compartment temperatures Wait until the analysis temperature is stable as shown in the status bar before starting the run 5 Prepare your samples and reagents in the microplate and or reagent rack Note the rack positions and volumes of samples that you prepare there is no software support in manual run for identifying samples or monitoring the volume of liquid in the autosampler positions You insert the samples as part of the starting procedure for the run You can also add samples during the run Biacore T100 Software Handbook 15 version AB Manual run 3 2 Starting a manual run Choose Run Manual Run to start a manual run LU Manual Run Lx Flow Reagent Rack 2 x F Flowrate E l min Flow path Reference Detection in flow cells 1 2 3 4 subtraction c E Flowpatht E Flowpatht 2 rone 7 C E Flow path 2 C B Flow path 3 4 none z c Fewpah3 E Flowpath1 234 2143 gt C E Flow path 4 Help Eject Rack Choose the initial settings for flow rate and flow path with reference subtraction You can change the flow rate at any time during the run You can change the flow path at any time during a cycle the available options are restricted by the choice made when the cycle is started Choose the rack and microplate settings These will apply throughout the run and cannot be changed Click Eject Rack to eject the rack tray so that you can load your sampl
194. taining recovered analyte is deposited in the target position where it mixes passively with the deposition solution 6 Steps 2 5 are repeated for the specified Number of repetitions This increases the yield of recovered analyte without requiring additional commands The same target position is used for recovered analyte from all repetitions Note that only one aliquot of deposition solution is used regardless of the number of repetitions Notes Methods that include the InjectAndRecover command require a sample and reagent rack type 1 and cannot be used with microplates see the Biacore T100 Instrument Handbook for rack details The contact time for sample flow rate and number of repetitions determine the total injected volume for both sample and recovery solution You may need to adjust one or more of these parameters if the method does not pass verification Regeneration command This command is intended for injection of a regeneration solution following the sample injection Check High Viscosity Solution if the regeneration solution has a relative viscosity higher than about 3 corresponding to about 35 glycerol or 40 ethylene glycol at 20 C This will adapt the solution aspiration and injection procedure for higher viscosity The injected solution contact time and flow rate can be set as variables 76 Biacore T100 Software Handbook Version AB Methods Sample command This command is intended for injection of sample contai
195. ter from the list If you attach a parameter to Keyword the initial parameter value will be set to the value of the keyword with the same name as the parameter Check Allow negative value if the parameter can be below zero Enter a description of the parameter for ease of identification If you have only used single parameter names as opposed to expressions for the rate constants and properties you can click Rate equations or OK as a shortcut to defining parameters The software will then enter suggested definitions for all undefined parameters This shortcut cannot be used if you have entered expressions Biacore T100 Software Handbook 157 version AB Kinetics and affinity analysis In the Report panel define the parameters you want to appear in the Report tab of the results Report parameters are defined by a name that may be chosen freely and a value that is entered as a parameter or expression containing parameters Click Rate Equations to display the equations generated by the software CE M d dt tcf 1 3 Conc A ka amp B kd AB B 0 RMax dB dt ka A B kd AB AB O 0 d4B dt ka A B kd AB You can select the equations in the display and click Copy to copy the equations to the Windows clipboard Use this function and paste the equations in to e g Wordpad to print a copy of the rate equations 9 4 2 Equation models for kinetics Models for steady state affinity evaluation are entered
196. ter keywords 102 filtering report point table 120 fit by color 117 fitting curves to plots 117 fitting function 125 fitting model 134 Biacore T100 Software Handbook Version AB Error Reference source not found fitting parameters 134 136 137 flow cells 1 flow path for buffer scouting 45 for capture 24 in wizards 24 manual run 16 18 method runs 82 flow rate effect on binding rate 59 manual run 16 18 range 26 folder for templates and methods 14 21 63 four parameter fit 117 125 free energy change 161 G general command 75 global parameters 137 140 gridlines 13 98 GxP compliance 2 H heat capacity 162 heterogeneous analyte 147 heterogeneous analyte kinetics method example 88 heterogeneous ligand 149 high performance 77 I if then command 73 immobilization 34 results 38 immobilization methods 35 immobilization pH scouting 32 import program 173 importing data to sample tables 172 importing rack positions 30 173 importing sample tables 48 53 56 83 importing wizard templates into Method Builder 63 initial values 157 inject and recover 75 inject sample manual run 18 injection parameters 25 injection sequence 23 injection volumes 26 instrument preparations for manual run 15 interaction models 154 Biacore T100 Software Handbook 181 version AB Index 182 K keyword table 101 kinetics 133 predefined models 144 kinetics control experi
197. ting fits 134 adaptive methods 73 adding keywords in evaluation 103 adjustment for molecular weight 114 affinity 133 application wizard 54 affinity in solution 129 calibration curve 169 evaluation 167 experimental design 167 method example 87 requirements for evaluation 86 168 variables 79 aim for immobilized level 36 37 aligning sensorgrams 111 analysis temperature 1 27 71 thermodynamics 58 analysis temperature after run 66 analyte 155 analyte concentration 78 analyte molecular weight 56 78 adjusting response 114 anchor 30 append file 96 128 137 application wizards 5 21 applying solvent correction 105 assay methods 63 assay setup 24 assay step name 68 assay step purpose 68 101 assay steps 64 67 analysis temperature 71 buffer change 71 recurrence 70 replicates 69 run order 69 assays steps connecting to cycle type 69 auto scale 10 automatic positioning 29 avidity 146 Biacore T100 Software Handbook 177 version AB Index 178 B baseline for report points 80 101 120 in regeneration scouting 42 in sensorgram display 11 plot 99 Biacore methods 87 BIAnormalizing solution 27 binding analysis 47 results 49 binding capacity 155 binding levels 99 binding to reference 99 bivalent analyte 146 blank immobilization 36 blank subtraction 113 buffer change 71 buffer names 67 buffer scouting 45 results 46 buffer selector 1 bulk refractive index contrib
198. tion first exports the rack positions table to a temporary tab separated text file which is processed by the import program specified in the Tools Preferences dialog The output of the import program is then imported to the Rack Positions table replacing the existing positioning information See Appendix A for more details Import Positions Imports positioning details from an external file Details of the import settings and file format are described in Appendix A Biacore T100 Software Handbook Version AB Application wizards Export Positions Exports the data in the positioning table to a tab separated text file See Appendix A for details of the exported file format Print Rack Positions Prints a copy of the rack positions diagram and table Print Wizard Template Print Method Prints a copy of the currently open wizard template or method 4 2 6 Prepare Run protocol This dialog box allows you to enter a run protocol to provide instructions to the user when the run is started A suggested general protocol is provided Ta PositionTest Prepare Run Protocol ni xj Tahoma z fio x B z u Prepare Run Protocol Make sure the correct sensor chip is docked e Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup Vials should be sealed with rubber caps and microplate with adhesive foil Place the buffer s on the left hand tray and insert the correct tubing s se
199. tion period No Cone varies by cycle MW varies by cycle Repott points a Expand All Collapse All This screen provides a summary of the method The left panel shows the assay steps in the method see Section 5 5 Click on an assay step to show the settings for the step and the details of the cycle definition see Section 5 6 in the panels to the right The cycle definition is listed as a series of injection commands to see command details expand individual commands by clicking on the marking or use the Expand All button to expand all commands in the panel This screen is for information only settings cannot be changed here Biacore T100 Software Handbook 65 Methods 5 4 General settings 66 Tas KineticsDiffBuffers Main ioj xj At start GeneralSettings Concentration unit Data collection rate Sample compartment temperature Vary with analysis rm zi fi te p we Cycle Types Sample amp Alter run _ Miscellaneous settings an Speciy analysis IET Setting Value Verification temperature after run Buffer A name 10 mM Tris NaCl The name of the buffer in position A Buffer Brame HBS EP The name of the buffer in position B Buffer C name PBS The name of the buffer in position C Buffer D name Empty The name of the buffer in position D The settings that are specified here are At start Concentration unit This setting defines t
200. tions Temperature 5 bal om times As entered 1 23123 Bin Order 11 2233 C Random This screen determines the main structure of the method in terms of assay steps Steps at the top level i e not indented or marked with the symbol are executed in the order given Nested steps marked with the symbol are executed within the context of the level in which they are placed as specified by the settings for Recurrence To create a new assay step click New Step The step will be created with default settings at the end of the current method Move the step in the method with the Move Up Down buttons until it is in the required position in the method You can make a copy of the currently selected step with the Copy Step button The Simulate Cycle Run List button beside the method summary panel allows you to check the number and order of cycles for the method as defined Biacore T100 Software Handbook 67 Methods xi Assay step name Assay step purpose Cycle type Startup Y Startup Startup Sample analysis Buffer A 20 Startup Startup Sample analysis Controls buffer A 2 Startup Startup Sample analysis DMSO buffer A 1 Buffer A Sample Sample analysis purer B 20 Buffer A Sample Sample analysis Controls buffer B Buffer A Sample Sample analysis DMSO buffer B 1 Buffer A Sample Sample analysis Buffer A Sample Sample analysis Buffer A Sample Sample analysis Buffer A Sa
201. to reveal trends in the regeneration performance Use variants of the same kind of regeneration conditions e g different pH values or different concentrations of ethylene glycol within the same run Results are most easily interpreted if you use a new flow cell or sensor chip for each kind of regeneration condition that you test so that the outcome with one kind of condition is not affected by the history of exposing the ligand to another You may choose to lock the solutions or the contact time used for regeneration tests so that all conditions will use the same setting for the locked parameter Do not vary both the solution and the contact time at the same time the results may be difficult to interpret clearly Check High viscosity solution s if any of the regeneration solutions tested has a relative viscosity higher than about 3 corresponding to Biacore T100 Software Handbook 41 version AB Application wizards 42 about 35 glycerol or 40 ethylene glycol at 20 C This will modify the injection procedure for better handling of viscous solutions 5 System preparations Check the System preparations options as required see Section 4 2 4 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 5 then save the wizard template and start the run 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 6 This text will be displayed at the st
202. ts menu options Notebook Right click menu options in the notebook represent standard Windows editing functions Biacore T100 Software Handbook 13 version AB Control Software general features 2 4 File storage 14 2 4 4 Wizard templates and methods Wizard templates are saved in files with a file name extension bw where represents an abbreviation that identifies the wizard e g a wizard template for concentration analysis has the extension bwConc Methods are saved in files with the file name extension Method Note The extension will not be displayed if the default setting Hide file extensions for known file types is selected in the Windows Explorer folder options Turning this setting off can help you to identify file types in dialog boxes Templates and methods may be saved in any location A folder structure under the default location as specified in Tools Preferences is however recommended since files in this location are handled preferentially in the Open New dialog boxes for wizards and templates see Section 4 1 1 Click on the browse button to change the folder setting 2 4 2 Result files Results are saved in files with the file name extension blr Result files from wizard or method based runs contain a copy of the wizard template or method as well as the results of the run Biacore T100 Software Handbook Version AB Manual run 3 Manual run Manual run allows you to control a run intera
203. tual temperature at the start of an assay step does not match the setting for the step the system will wait until the setting is reached Buffer Select the running buffer to be used for the assay step The default buffer is A corresponding to buffer bottle and tubing A on the instrument Te BufferChange Main EI 1 Create a new cycle type or select an existing from the list Cycle type description Cycle types currently in method Sample analysis Solvent correction Verification 2 Insert the required commands 3 Configure settings for each command Commands Report Points Insert Command Remove From Cycle Move Up Move Down Sample 1 Regeneration 1 Settings for Sample 1 Type Method Variables Evaluation Variables Solution Ig Contact time s IZ Dissociation time s C Flow rate u min Solution Is variable Contact time Is variable Dissociation time Is variable 30 min 1234 Jm Flow rale Flow path T Predip Cmi o S Fraction T 2 of mi solution FF Steblization period aterm 0 a o T Extra wash after injection with IT Stabilization period Cycle types define the detailed sequence of operations to be performed in each assay step The top panel in the work area lists the cycle types currently available in the method Use the New button to create a new cycle type Mark a cycle type and click Delete to remove the cycle type from
204. ult file in the evaluation software use the File Open menu option You can open result files from the control software file extension blr and saved evaluation sessions file extension bme If a file is already open in the software opening a new file with File Open will automatically close the first file Opening a file automatically creates a Sensorgram item and a number of default plots according to the content of the file To open multiple result files in the same session use the File Append file option You can only append result files not evaluation sessions Appending a file to a session will delete all the user defined evaluation items in the session 6 3 Common display functions 96 6 3 1 Zooming the display To zoom a display window drag with the mouse over the area you want to enlarge To restore the previous zoom level double click anywhere in the display window except on the axes or legend or select Unzoom from the right click menu Displays are normally rescaled automatically whenever you change the displayed data To keep the current zoom setting when data is changed check Zoom lock in the display window Biacore T100 Software Handbook Version AB Evaluation software general features You can also set the display scale with the Scale option from the right click menu The display is not rescaled automatically if the scale has been set to specified values 6 3 2 Right click menus Right click in display
205. urpose list Report point values are calculated for all cycles in the selected assay steps Editing and deleting report points Select a report point in the list in the Custom Report Points dialog and click Edit to edit the report point definition or Delete to delete the report point If you delete or change the name of a report point that has been used in a custom plot the corresponding plot window will be empty 6 6 Keywords Keywords are assigned to cycles when the run is performed and are then used for identification and evaluation purposes Keywords are created automatically for wizard based runs and may be defined in the method for method based runs and include e automatically generated identifiers such as cycle number or assay step purpose e method variables and predefined evaluation variables such as sample name concentration and molecular weight e user defined variables see Section 5 6 2 You can add and remove user defined keywords in the evaluation software and edit the contents of certain keywords Choose Tools Keyword Table to open the keyword table When you edit the keyword table all user defined items in the evaluation session will be deleted Save the session before editing the keyword table if you do not want to lose your work Biacore T100 Software Handbook 101 version AB Evaluation software general features 102 amp Keyword Table xj Cycle Assay Step Pu
206. ution 156 C calculated concentration 12 6 calculated concentrations 125 calculated parameters 158 calibration curve 51 124 calibration curves 169 caption 97 capture 23 73 flow path 24 plot 99 carry over control 73 90 plot 99 chack kinetic data 141 changing rack positions 28 chip properties 8 10 38 chip type 35 chi squared 139 commands in Method Builder 72 commands in manual run 17 competing analytes 147 complex 156 concentration analysis application wizard 49 combined result sets 128 evaluation 123 requirements for evaluation 85 123 concentration series 130 concentration unit 66 102 Biacore T100 Software Handbook Version AB Error Reference source not found conditional command 73 conformational change 151 connect to cycle type 69 constants 140 contact time at low flow rates 26 control experiments 59 results 61 control samples binding analysis 48 concentration analysis 52 125 kinetics affinity 56 plot 99 control software Edit menu 9 File menu 7 Run menu 11 screen regions 6 Tools menu 12 View menu 10 copy graph 13 creating a method 63 creating a wizard template 21 creating fitting models 153 curve fitting 117 138 custom immobilization methods 37 custom report points 80 100 cut data 113 cycle manual run 19 cycle run list 83 cycle types 64 71 D data collection rate 66 data presentation 109 deleting report points 101 deposition soluti
207. ve is used 8 2 Evaluating concentration analysis To evaluate a concentration analysis run open the run and click Concentration Analysis in the evaluation toolbar or choose Add Concentration Analysis from the Evaluation menu The evaluation dialog is divided into three tabs for calibration curves control samples and unknown samples respectively Choose the appropriate settings for the calibration curves and click Finish to complete the evaluation There are no settings that can be changed for control samples or unknown samples Biacore T100 Software Handbook 123 version AB Concentration analysis 124 8 2 1 Calibration curves Ws Concentration Analysis Create f x Calibration Control Samples Samples m Calibration curve settings Flow cell Fc 1 vi Report point stability v Response type Relative response Fitting function a Parameter Calibration table Calibration curve Curve Cycle Conc Response Calc Conc cv s cave 1 inl ng ml RU ng ml 9e RU Biotin 1 9 1 02 1380 5 0 9948 1600 4 Avg 1380 5 0 9948 NJA 1400 4 1 8 2 56 1298 6 2 632 Avg 1298 6 2 632 NJA 1200 4 1 7 6 4 1142 2 6 296 8 Avg 1142 2 6 296 N A i 1000 4 i IE 1 6 16 861 7 16 13 i ri soo Avg 861 7 16 13 NJA 1 a 1 5 40 552 0 39 82 ST Avg 552 0 39 82 NJA 400 4 Parameters for curve 1 200 t t t t t 1 Rhi 3362 Hlo 1432 A1 2142 A2 1 051 20 0 20 40 60 8
208. ware Handbook 135 version AB Kinetics and affinity analysis The detailed results are presented on three tabs e Report shows selected parameters and calculated values The contents of the Report tab are defined in the model Global parameters are listed on a single row at the top of the table and local parameters are listed on one row for each curve e Residuals plots the difference between the experimental and fitted curves for each point in the curves Use this display as an aid in judging how closely the results fit the experimental data e Parameters shows the values for all parameters in the fitting equations Kinetic constants obtained from the fitting procedure are only significant if the observed binding is not seriously limited by mass transport of analyte to the surface see Section 9 3 For 1 1 fitting results you can check whether mass transport is limiting or not using the Check Kinetic Data function as described in Section 9 2 3 Affinity results For affinity determination the reported Kp value is marked on the plot as a vertical line for a 1 1 interaction Kp is the same as the analyte concentration at a response equal to half Rmax If the reported value is higher than half the highest concentration used this line will be shown broken in red as a warning that the value may be unreliable because the plot does not flatten out sufficiently EB Kinetics Affinity Fit Affinity Create x
209. wever important to remember that the name is for documentation from the user s perspective and may be chosen freely while the purpose has significance for the step properties and for evaluation of the run and must be chosen from the predefined list Connect to cycle type Each assay step is connected to one cycle type which determines the detailed operation of the step see Section 5 6 Choose the cycle type from the list of types available in the method 5 5 2 Number of replicates Assay steps can be set to run in replicate which means that all cycles in the assay step will be repeated the specified number of times The order in which cycles in the assay step are repeated can be specified As entered performs all cycles in the step once then repeats the step until the number of replicates is reached this is represented as 1 2 3 1 2 3 to illustrate the order of 3 cycles in a step repeated twice Biacore T100 Software Handbook version AB 69 Methods 70 Order performs the first cycle in the step for the specified number of replicates then the second cycle and so on represented as 1 1 2 2 3 3 Random randomizes the order of the cycles within the step until all cycles have been executed the specified number of times Any steps that recur in a replicated step are performed at the specified recurrence interval see below within the total number of cycles in the replicates of the step The number of replicates for the re
210. which returns a non zero value for subset n only These in parameters are listed on the Parameters tab in the results 9 2 Curve fitting principles 138 With all kinetic and affinity analysis it is important to remember that the results obtained represent the results of fitting the experimental data to a mathematical model and that obtaining a good fit is not in itself evidence that the model describes the physical reality of the interaction The fitting procedure does not have any knowledge of the biological significance of parameters in the model equations and it is wise always to examine the results obtained for reasonableness of the values obtained In addition any mechanistic conclusions drawn for Biacore T100 Software Handbook Version AB Kinetics and affinity analysis the interaction from fitting results e g concerning multiple interaction sites or conformational changes should ideally be tested using independent techniques 9 2 1 Fitting procedure Kinetic parameters are extracted from experimental data by an iterative process that finds the best fit for a set of equations describing the interaction The equations are created automatically from the definition of the interaction model The fitting process begins with initial values for the parameters in the equation set and optimizes the parameter values according to an algorithm that minimizes the chi squared value for the fitting Chi squared is a measure of the averag
211. xpressions for the reactant properties Note Numbers are used as part of the identifier not in the conventional chemical sense of stoichiometry Thus a complex named AB2 does not imply two molecules of B binding to one of A 154 Biacore T100 Software Handbook Version AB Kinetics and affinity analysis Analyte The analyte is injected in solution at a constant concentration and has the properties listed below Analyte is usually denoted by the letter A Concentration Injected concentration in molar units Injection s Start and stop times for the injection Mass transfer Check this box to include a mass transfer term in the fitting and enter a parameter name or expression for the mass transfer constant Molecular weight Check this box and enter a molecular weight if required This information is used to calculate relative response contributions for heterogeneous analyte models it is not used for conversion of weight based to molar concentration units this conversion is performed if necessary in the sample table Number of blocked sites Check this box and enter a number if binding of one analyte molecule sterically blocks additional ligand sites This box should normally be left unchecked Ligand The ligand is immobilized or captured on the surface and has the properties listed below Ligand is usually denoted by the letter B Binding capacity Maximum analyte bi

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