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Biacore T200（Ver.2）取扱説明書 基本操作編

Contents

1. D w Sensor Chip i 1 P uu CM5 M n e A Biacore T200 9 169 9 Biacore T200 Evaluation Software
2. OFF TEL 03 5331 9336 AL ERIT 9 00 17 30 Q FAQ
3. Biacore T200 94 5 5 2 1 Toolbar Run Method amp x Menu bar Run Method Te Open New Method in Melhods And Templates Modified Biacore Methods Biacore Methods Open l Ta Open New Method Lookin Biacore Methods Name Ed Affinity in solution 5 Calibration Free Concentration E GST Kinetics Inject and recover E Kinetics heterogeneous analyte 5 L1 liposome capture ED LMW kinetics E LMW screen EANTA kinetics I Single cycle kinetics Type Method Builder Method Builder Method Builder Method Builder Method Builder Method Builder Method Builder Metho
4. RI R RI 0 Constant lt 1 ka ka Rn lt Rr
5. Bulk Effect ng ml ug ml Kp 1 10 10 8
6. Assay Steps Cycle types Cd Assay Steps Startup Samples Control Samples Cycle types Assay Steps Cycle Types Biacore T200 6 125 6 1
7. N g CM NHS N NHS j S S S S a I E HOD3EXS7U AK Um C 2509383 7388 IC d OBS UO PZJ bTEPFE amp IERKUC E iT ANKLE
8. 6 4 Excel Excel 2 Excel txt Next gt l Biacore T200 146 6 Te Method Builder Cycle run list Cycle Assay step name Sample 1 Solution Sample 1 Conc nM Sample 1 MW Da buffer buffer buffer negative control positive control sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 sample 1 negative control positive control sample 2 sample 2 sample 2 sample 2 sample 2 sample 2 v
9. HE 4 1 1 AES pH NaCl lt 2 M 10 mM Gly HCI HCI Phosphoric acid Formic acid 10 mM Gly NaOH NaOH Ethanolamine Ethanolamine HCI EDTA Surfactant P 20 Tween 20 Triton X 100 SDS Octylglucoside Acetonitrile 2096 DMSO 896 Ethylene glycol in HBS Buffer 5096 Ethanol 2096 Formamide 4096 ZERI Guanidine HCI Urea Biacore T200 4 47 Toolbar Start Manual run Ep Menu bar Run gt Manual run Te Manual Run Flow Flow rate 30
10. CM CM 0 5 pH NNN NN pH Immobilization pH Scouting pH C pH
11. Biacore T200 18 2 2 1 1 Inject command Le Menu bar Commands Inject Inject Vial well position A1 A1 Contact ime 6 Minimum required volume in vial well for this injectio Vial well position contact time Inject 98 Biacore T200 Control Software manual bir UE IEG File Edit view Commands Run Tools Help B8x Hx lc 7 E i Cyde 1 Curve Sensorgram Fc 1 exvsrssi3ilcoii RU C Lock scale lu scs vn Vial well position Contact time Minimum required v 8 90 Fc Time Window A amp bsResp SD LASO Keywords in cycle 1 Value Flow 30 Flow Path 1 Online COM1
12. Finol Bound NHS Final Bound _ Biacore T200 3 41 3 1 3 Aim for Immobilized level 3 2
13. Reagent rack Type 1 15m 11 mm x 20 Reagent rack Type 2 16 mm 4m x9 15 mm 4 ml x94 7 mm 0 8 ml x24 Sample and reagent rack 16 mm 4ml x9 15mm 4 ml x94 1 5 ml 11 mm x 24 7 mm 0 8 ml x45 Rubber caps type 3 Rubber caps type 2 Rubber caps type 5 BR 1005 02 LJ BR 1004 11 cy BR 1006 55 R 7 mm Plastic Vials 1 5 ml Plastic Vials 16 mm Glass Vials 15 mm Plastic Vials BR 1002 12 ES BR 1002 87 Dh 1002 09 BR 1006 54 J Rack tray 96 well 384 well F x Microplate 96 well BR 1005 03 Microplate Foil 96 well 28 9758 16 Microplate 384 well BR 1005 05 Microplate Foil 384 well BR 1005 7 7 Biacore T200 1
14. 20 Sample compartment 20 Biacore Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BIAdesorb solution 2 50 mM Gly NaOH pH 9 5 Tools gt More Tools Maintenance Tools gt Desorb Start This procedure removes adsorbed material from the Flow system Total run time is about 20 minutes NOTE Uze the Maintenance Chip fer this procedure The surface on other sensor chips may be damaged by the solutions used Din nat run this procedure below 20 C Next gt liesnrh Required solutians from Maintenance Kit Bl amp desarb salutian 1 Bl amp desaerb solution 2 Next gt BIAdesorb solution 1 BIAdesorb solution 2 L Start Desorb Standby_flow
15. Residuals Y B Z 1 lt 2 RU Residuals for a poor fit RU Residuals for a good fit RU Biacore T200 5 75 Report Kinetics Affinity Fit Kinetics Create Lurve Fc 2 Ligand antibody 1 ug ml pH Sample antigen Temperature 25 LC Add Fit Current Filz Madek 1 1 Binding F 1 1 Bindim me ualtu Control Report Residuals Parameters Curve ka r Ms kd 1 s KD M Rmax RU Conc M tr Flow ul min kt RU Ms RI RU Chi RUP U 1 531E 5 D D02610 1 705E 9 7 178E 7 1 1 E 9 2 231E 8 0 05105 2 200E 9 2 231E 8 0 2011 4 300E 9 2 231E B 0 1965
16. hug m Biacore T200 46 4 30 1
17. Assay Steps New HPne J Purpose Cycle type 6 3 Move Up MoveUp Move Down Move Down Delete Dete 2 Base settings Name Purpose Biacore T200 6 131 Purpose d Evaluationsoftware
18. 060327 Thermo Position uc Content Sample 1 48 Negative control Control sample 148 Negative control Control sample e 148 Negative control Control sample 148 Negative control Control sample i 148 Negative control Control sample e CX i 148 Negative control Control sample 1 t i 148 Positive control Control sample OQ OO Lis ug Ii EE i Ositive control Control sample 96 Deep Well Microplate i 148 Positive control Control sample i 148 Positive control Control sample i 148 Positive control Control sample E 9 Q Q Q Q Q Q Q i 148 Positive control Control sample 00000000 148 Andyte Sample i 148 Analyte A Sample 100000000 E AOBRRRDBRBBBB5ll5l1 g 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 440 ne Menu 7j 5 Automatic Positioning l T Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orienta
19. ea Biacore T200 16 2 Save Results From Run hs Savein T100manual v 0O f emn My Recent Documents My Computer Mu Network Save as lype Result file bir w C NB Usersv Sae l 18 Biacore T200 Control Softwaree manual blr bz Fle Ed iew Commands Run Tools Help id FE j Em E mA b e Cycle 1 x Curve Sensorgram Fc 1 a IZOR 314 cJ M 0 5 10 15 20 25 40 Time C Lock scale Fc Time Window amp bsResp SD LASD Slope RelResp Baseline ld Keywords in cycle 1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 C Running manual run Sample compartment temperature current 25 C set 25 C Run Lime 1 min Biacore T200 2 17 E 2 2
20. 5 10 10 30 Biacore T200 5 57 5 1 1 Toolbar Run Wizard 7 Menu bar Run gt Wizard m Open New Wizard Template GI Surface Preparation Immobilization pH Scouting Immobilization G Assay Development i Regeneration Scouting Buffer Scouting Surface Performance C Control Experiments i Kinetics Linked Reactions Look in G3 Methods And Templates Name Kinetics Mass Transfer E Assay 2 Binding Analysis Concentration Analysis Thermodynamics Immunogenicity i Immunogenicity Screening PA Immunogenicity Confirmation Immunogenicity Isotyping Jus Heb ese_ Assay Kinetics Affinity New Methods and Templates 2
21. Fit Rmx Rmax Rmx Rm MW analyte Rm Xqnalyte RMOXcontrol X MW control Biacore T200 5 91 Steady State Affinity Constant Rmax Multi Site 2 Rao Rao Rmaxr Rm Rmoa CR nox m CR Ran Kot C Kp C max offset Kinetics Affinity Fit Affinity Create Curve Fc 4 3cor Ligand H A Sample Furosemide Temperature 25 C Add Fit Current Fits Model Steady State Affinity Steady State Affirity Description 525 B Concentration Report Paramet
22. J 73 24 FX Kp M 1 Kp KA 1 M A B AB Ko A B AB BBCk Biacore Ko ka ka Ko Ms REBEXRIEEAEEX
23. Ese RE View Base Line F9 0 Biacore T200 52 4 4 4 Application Wizard Assay Development Regeneration Scouting A ZIODFBESR C
24. include Biacore T200 5 69 ES Kinetics Affinity Select Curves Create Select evaluation mode Single mode C Batch mode Curves Cave Fos21 v gend antibody TOugimli v Sample jesse dede Cvde mij Gm D n nM l min s s 1 1 30 2g 30 4 3 30 5 30 1 30 30 C Zoom lock 5how concentration series Show blankis Show average blank s Multiple Hmas Adjust Injection E vents Next gt ES Kinetics Affinity Select Data Create Curves Curve Fc 1 Ligand antibody 10ug mlpH5 Sample antigeni Temperature 25 C nM pl min s s b5 Fe 2 1 1 1 3 amp Fc z 1 30 y Fce z 1 1 3 3 B Fc2z2 1 B 3 8 Fc 2 1 1 3 Blank Subtracted Sensorgrams E Zoom lock E Biacore T200 70 5 0
25. Toolbar Eject Rack H 3 Eject Rack Tray Hack Tray Ejected Click OK to retur the rack tray In the sample compartment Time to auto close 50 Eject Rack Tray 60 OK Biacore T200 12 1 E 1 10 Y
26. Commands Regeneration Remove Biacore T200 5 99 Startup Te Method Builder Main Cycle types Description of selected cycle type This cycle is used in start up steps Sample General Settings ES Contains injection sample and regeneration The sample is of the type low sample consumption not single cycle kinetics to save time during start up procedures The normal sample cycle could be used Assay Steps as a start up cycle also Cycle Types Variable Settings Commands ReportWPoints Verification N x v Setting for Sample 1 3 De Low sample consumption v Method Variables Evaluation Variables tT Sample solution ls variable Set property as variable none M EET wee le pe Dissociation time s errr Flow rate l min Elow path C Predip a Mix with Type Low Sample consumption contact time 60 s Dissociation time 60 s
27. R RI 0 Constant lt 1 ka ka Rn lt Rr RU Ra
28. Next gt Te Kinetics Affinity etup Conditioning Bun conditioning cycle Contact time m s Mumber of injections Startup Run startup cycles Solution buffer Number of cycles Solvent correction Bun solvent correction Number of injections Repeat after sample cycles Startup Solution Biacore T200 5 59 Number of cycles 3 Next gt Te Kinetics Affinity Injection Parameters Sample Contact time 120 s Flow rate pl min Dissociation time 120 s Estra wash after injection with Regeneration Solution Gl HCI pH2 5 C High viscosity solution Contact time s Flow rate umin Stabilization period s Sample contact time 120s Flow rate 30 ul min Dissociation time FE SEE ST fes 120 s Regeneration
29. RU RU Biacore T200 4 51 New Cycle C23 1 End Manual run eb ala Menu bar Commands gt End Run Standby flow 4 3 Tool bar Reference Line 3z zEbasevva View Reference Line
30. New chip Dock Chip Properties Reuse chip id Dock Biacore T200 8 1 1 7 Series S Sensor Chip CM5 3 BR 1005 30 Series S Sensor Chip CM4 3 BR 1005 34 Series S Sensor Chip CM3 3 BR 1005 36 Series S Sensor Chip C1 3 BR 1005 35 Series S Sensor Chip CM7 1 28 9538 28 DNA Series S Sensor Chip SA 3 BR 1005 31 Biotin CAPture Kit Series S 1 8 28 9202 34 JRIKE9 7 Series S Sensor Ch
31. After run Biacore T200 130 6 Assay steps T Method Builder Main Startup General Settings x Delete Startup LMW kinetics 3 times as entered Co z Assay Steps 2 Sample Sample LMW kinetics 1 time as entered Cycle Types Move Up Solvent correction Variable Settings t Solvent correction Solvent correction 1 time as entered Before after every 20 cycles I Move Down Verification Control sample t Control sample LMW kinetics 1 time as entered Before after every 10 cycles SetupRun Cycle Run List Assay step properties Base settings Recurrence Name Startup C Repeat assay step within Purpose Startup he Connect to LMW kinetics Distribute occurrences evenly cycle type i BE din Assay step preparations 5 Number of replicates Temperature times Buffer s entered 1 2 3 1 2 3 Drder 1 1 2 2 3 3 O Random Assay steps 5
32. Evaluation purpose Evqluation software _ User defined variables FO Add Evaluation purpose Sample Evaluation purpose 7 Kinetics Affinity Thermodynamics Concentration Affinity in solution Kinetics Heterogeneous analyte Calibration free conc General Biacore T200 140 6 Report Points Er Report Points me sec getore aner Statu iniect widow Rosin E bas
33. Toolbar Run Method amp Menu bar Run Method Te Open New Method ook in Methods nd Templates Method Builder 4 2 2008 Method Builder 9 13 2007 C iee Come wowmewaseedewae mee Ln ten Methods and Templates 7 Browse Open Biacore T200 126 6 T Method Builder Main Assay steps General settings Concentration unit nM General Settings Startup Data collection rate 10Hz j Startup LMW kinetics 3 times as entered Sample compartment temperature 25 C Detection Dual Assay Steps A Sample Cycle Types Sample LMW kinetics 1 time as entered Settings for assay step Startup Temperature 25 C Variable Settings Solvent correction Buffer t Solvent correction Solvent correction 1 time as entered
34. 0 6 141 6 3 Solvent correction Cycle types New General Settings Assay Steps C H8 Cyce 5 Cycle types Thermodynamics Conditioning Solvent correction G Commands Report Points Cary ower control Solent corecton Inject amp ndR ecover Commands Solvent correction Insert Ene Solvent c
35. Copy Graph Paint WordPad Biacore T200 5 87 5 1 3 Evaluation k E L3 a MfS x Edit PAGE Curve Name Fc 4 3 P P gt Sensorgram 4 All sensorgrams RU k 25 Plat 8800 O Zoom Loc Baseline Sample a Binding level 8600 Binding stability Binding to reference Controls binding 8400 Controls stability Report Point Table mm Report Point Table 8200 Conditioning ll 8000 Control Sample 7800 Sample mmy p E perd 3 Solvent correction 7600 Il Startup 7400 eT 7200 i 7000 6800 Time Keyword table Tools Keyword Table
36. Temperatur t 79 3wv7 Sample compartment temperature Run Biacore T200 1 5 1 2 1 2 1 Insert Chip Biacore T200 Insert Chip e Mew chip Q Reuze chip Mew chip Chip 1d d80415 0213 12114 Insert Chip e Mew chip Q Reuze chip Mew chip Chip type LM Chip 1d Chip lot na Series S CM5 New Chip Reuse Chip Chip type 7 l Insert Chip New chip Reuse chip New chip mwe mg v Chip id 080415 0213 12114 Chip lot no optional 10119353 Chip id
37. Verification Biacore T200 144 6 T Method Builder Main Verification results General Settings Variable Settings Veification The method has been verified and can be used to setup a run SeupRun The method has been verified and can be used to set up arun Setup Run Te Method Builder Detection Detection Flow path Flow path Next l Biacore T200 6 145 Ce Method Builder Variahles Assay steps Startup Sample Control sample Variable values for Assay Step 5 ample ee QR T S m AC I 3 Epi na 1 95 7 81 3l 25 125 IBN 2000 bal Sample amp Assay Setup Define all values at run time
38. 7 mm 58 ul ls variable Cd s S ul min Detection Dual First 2 1 1 4 3 3 Predip Mix with 6 137 Second 2 1 2 4 3 4 Both 2 1 1 2 4 3 3 4 Detection Multi
39. Custom Methods Amine Ta Custom Methods Methods I Aldehyde Te mine DA Ligand thiol 1 3 Maleimide Tr Surface thiol Biacore T200 36 3 Copy Methads Copy of Amine Methods l Method name Command Solution Contact Time s Flow Rate ulimini ps PRE CQ Specified in Immobilization Setup nia PISIMJECT EDT NHS 50 50 420 10 3 WASH Ethanalamine r3 LIGAMDIMJECT Specified in Immobilization Setup P IMJECT Ethanolamine 420 in Edit EDC NHS Mix amp Inject Solution EDC Mix withe NHS Cancel Fraction of mis with solution TT Contact lime s Elow rate ulmin OK het
40. Fc1 Fc3 Method Amine Ligand NHS 10 ul min 30 7 Aim for immobilized level Specify contact time and flow rate U 292 F OR DUBSHeSI CL 738 ec T8AE UL C lSXE4G C ee d Blank Immobilization NHS Specify contact time and flow rate 420 S 10 l min Next gt 3 2 Specify contact time and flow rate 7
41. Lo All sensorgrams 44 Curve Name Fc 2 rj Assay Step Purpose Overlay rj Cycle Overlay RU Sensorgram Zoom Lock Biacore T200 174 9 9 3 2 Sensorgram window _ ect Tools Color By gt Sample RU Sensorgram Zoom Lock 10700 a2micro high2 a2micro high1 a2micro low2 a2micro low1 9700 Tools gt Report Point Id and Marker RU Sensorgram T C Zoom Lock Gi 1230 s E m n 0 c 1225 s amp End mi gt 1220 E c 5 amp 1215 s 7 B ke E m E B Fk E 5 p n 1200 50 100 150 200 250 id Biacore T200 9 175 9 3 3
42. Open Browse Open Chip type Flow cells per cycle Fi Immobilize flow cell 1 Flow cell 2 L1 l Immobilize flow cell 2 Flow cell 3 L1 amp Immobilize flow cell 3 Flow cell 4 Fi BI Immobilize flow cell 4 Custom Methods Chip type CM5 Flow cells per cycle 1 2 4 Biacore T200 3 35 Flow cell 4 C3 Aim for immobilized level Ligand Protrein amp 20ug ml nH5 Dilute ligand Specify contact time and flow rate Contact time s Flow rate 10 mm C3 Blank immobilization Flow cell
43. Start 72 5 All programs Biacore Biacore T200 Control Software 1 4 8 Biacore T200 Control Software Immobilization of PrteinA bir ibg Eile Edit View Run Tools Help ERMINE Immobilization of PrteinA blr ER iacore T200 mmy iz File Edit view Tools Help X Menu bar Es Time Information E WES EENE 0 0 Timestamp 5 8 2008 1 38 13 PM Cycle 1 F20N 50000 0 0 Temperature 25 00 C 00 Data collection 1 Hz Event log 39 5j 0 0 Set Sample Temp 25 C 48000 4 0 0 Use Buffer A i 0 0 Actual Sample Temp 25 C 0 0 Flow 10 l min 46000 7 0 0 Set Temperature 25 C 1 0 0 Flow E m 44000 4 0 0 Dega 11 2 Transfer a 62 yl R2B1 R2B3 552 Transfer Ready E V e n t O g 42000 552 Wash Start Sensorg ra m ji 857 Wash Ready 2 953 Transfer Start 52 pl R2B2 2 R2B3 10 1380 Transfer Ready 1 1380 Mix Start 111 pl R2B3 window snl oe Oycle 1 x Curve Sensoreram Fc 4 X Curve Sensorgram Fc 4 ProtreinA 20ug ml pH5 Toolbar 251 3 Mix Ready 286 8 Inject Start R2B3 E 705 8 Inject Ready 70 yl 36000 3 719 9 Wash Start 750 4 Wash Ready EDC NHS 750 4 Wash R2B4 M 8053 Wash Ready 8405 Inject Start R2B5 12505 Inject Ready 70 yl 12735 Wash Start 1304 3 wash Ready
44. _ Immobilization pH Scouting 50 mM NaOH Biacore T200 28 3 Toolbar Run Wizard Menu bar Run gt Wizard 7853 Open Mew Wizard Template mm ourface Preparation o Immobilization pH Scouting Br Immobilization 7 Assay Development Regeneration Scouting Buffer Scouting Surface Performance Il Control Experiments Z Kinetics Linked Reactions Kinetics Mass Tranzfer Kinetics Affinity Binding Analvzis Concentration Analysis Thermodynamics EJ Immunogenicity Immunogenicity Screening Immunogenicity Confirmation Immunogenicity Izatvping Surface Preparation gt Immobilization pH Scouting New M
45. Edit Chip Information e E 3 i 3 ss sss ssa o es ssa sr es s lt es ssn ss 3 3258 3 3323 28 7735 ELI ssn 1 MAA Biacore T200 88 5 Toolbar p Kinetics Affinity 4 31 y pg A Surface bound amp Kinetics Affinity Select Curves Create Select evaluation made Single mode Batch mode Curves Curve Ligand Sample Furosemide w Temperature 5 vw CUCIEN Conc Flow Contact Time Diss Time isum pM Hl min s s 30 46 0 079 0 157 0 313 0 625 v L v L4 v La L L4 L4 Zoom lock Shor concentration series Show blank s Show average blank s Select Evaluation mode 1 Single mode Batch mode cd Bath mode 5 14
46. Menubar File gt Save 2 3 File Print OK Print Frnter Printer Sensargrarm C2 Mane Current Cycle Osme C3 Al cycles File Praperties Include event lag for cycles File Properties Wizard Template Method Wizard Results Sensorgram Current Cycle Range All cycles Include event log for cycles Biacore T200 3 23 90 eS fe BET CM5
47. Open Browse Open Te Kinetics Affinity Injection Sequence Detection Chip Elow path Chip type Capture SAMPLE REGENERATION 1 EEN Cary Over Biacore T200 58 5 1 Detection Flow path 2 1 4 3 Chip Chip type Capture 2 4 Sample Regeneration 1 or 2 Carry Over
48. Ctrl Breakl Biacore T200 Evaluation Software Biacore T200 66 5 5 1 2 Evaluatton Biacore T200 Evaluation Software Kinetics antigen antibody blr mij Uis r GM POT Help 3 RAUS MET NN Curve Name Fc 2 1 Assay Step Purpose verlay P Cycle Overlay J Sensorgram J All sensorgrams RU Sensorgram c3 Plot 3600 C Zoom Lock Baseline Sample Binding level Binding stability Binding to reference Report Point Table mm Report Point Table Conditioning Sample Startup 300 Time 5 6 Keyword table Tools Keyword Table
49. Di On o m EILAN ORAAL 91 5 3U 977 0u 3 JALAN OHE HITA Ab IC 60 Qe a5 B S B 8Bila 2 FRP CRUS VN i
50. Modification factor M 1 10 Biacore T200 5 77 ka k 1 10 Mbr Mies Compare to Response Suc RI
51. Standby flow Biacore T200 Evaluation Software HB Biacore T200 6 149 6 6 Control Sample R1A1 R1A12 12 060327 Thermo Position uc Content Sample 1 48 Negative control Control sample 148 Negative control Control sample e 148 Negative control Control sample 148 Negative control Control sample i 148 Negative control Control sample e CX i 148 Negative control Control sample 1 t i 148 Positive control Control sample OQ OO Lis ug Ii EE i
52. Flow rate 30 ul min Flow path Both Multi Commands Regeneration Remove Variable Settings Biacore T200 100 5 Te Method Builder Main Assay steps Define variable handling for each amp ssay Step Startup Define all values at run time EE O Define all values in method Vibes is s i Sample Solution SetpRun 3 Define all values at run time Define all values in Method lFERUIESXVvE amp SRSEICTLZUL Define some values in Method and others at run time Verification
53. RU Ra Rmo Rr Biacore T200 5 121 5 29 LA Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model ls 34 Binding HEBES Description Parameters i Delete Tools w ka 1 Ms kd 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms RI RU Chi RU U 1 531E 6 0 002610 1 705E 9 7 178Ec 7 1 100E 9 2 231E 8 2 200E 9 2 231E 8 0 4 300E 9 2 231E 8 8 500E 9 2 2
54. D 5 3 Menu gt Export Positions D Menu Simple Position Import Biacore T200 5 63 5 4 Control Sample R1A1 R1A12 12
55. t Sku J 9 Capture Sample Regeneration Sample _ Evaluation software Sample Types Sample solution Contact time Dissociation time Flow rate Flow path Biacore T200 Low Sample consumption 7 mm 28 High performance 7 mm 58 ul Single cycle kinetics 5
56. RU Adjusted sensorgram ic binding ability hinding ability lte xs nding nding bi Kstabilitystability zs Response 0 baseline S D c T m 100 50 0 50 100 150 200 250 300 350 Time 0 baseline Sensorgram window Te Tools Sensorgram Adjustment Biacore T200 9 179 Y Adjustment les s C5 Injection Event respanse D Enable Second Adjustment Wormalize CO Report Point response 10D Injection Event response 100 Enable Second Y Adjustment Normalize Injection Event response 100 Sample 1 stop OK RU Adjusted sensorgram 120 Response 0 baseline 100 Sample 1 stop 100 50 0 50 100 150 200 250 300 350 Time 0 baseline s 100 RU Biacore T200 180 9
57. _ 4 50 ml HBS EP 4 C e Sensor Chip i Lo CMS t Sensor Chip ju SET T C M 2 p Biacore T200 168 8
58. Evaluation 9 1 Start All programs Biacore Biacore T200 Evaluation Software S Biacore T200 Evaluation Software 5ensoraram Plot a4 Bar Chart AIK Evaluation Explorer n r7 Biacore T200 170 9 9 2 Cle File gt Open C BIA users C BIA users T200 demo File Binding Analysis OK YBiacoreT200 Evaluation Software KineticsAffinity 1 1 interaction blr s Curve Name Fcz2 1 e Assay Step Purpose verlay Ji Cycle Overlay ensorgram J All sensorgrams RU Sensorgram E gt Plot Baseline Sample Binding level Binding stability Binding to reference Report Point Table m Report Point Table C Zoom Lock Biacore T200 9 171
59. Menu bar Tools SetTemperature Biacore T100 Evaluation Software Eject Rack Rack Illumination Insert Chip Set Temperature Preferences More Taals set Temperature x Analysis temperature L Sample compartment temperature L 47 45 COD88BH CagxE LL C OK 15C Status bar temperature 27 Z7 TSESI2 m3 Lek d temperature 25C Biacore T200 1 11 1 2 4
60. 1 A Number of replicates Stt c As Entered Order Random Biacore T200 132 _ 6 6 1 1 Startup 1 Startup Thermodynamics 5 times as entered Control sample 1 Control sample Thermodynamics 1 time as entered Therma 1 sample Thermodynamics 1 time as entered Startup 1 Control Sample 1 Thermo 1 Recurrence
61. RU vs C ka ka Ko utbs RU RU 40x1 Sx RU 200x1 5x S 50 kDa Pyo4bkon cs 100 kDa 1 40 x 1 1 x 50 0007100 000 20RU 200 x 1 1 x 50 000 100 000 100 RU 207 100 RU Biacore T200 3 25 3 1 N g
62. Thermodynamics Kinetics Affinity Immunogenicity Single cycle kinetics 93 Calibration Free Concentration LMW screen Inject and recover Biacore T200 54 5 5 1 1 1
63. RI 4873 0 RU Residual Residuals Y B zt c1 2RU Chi U value 15 25 SE Standard error SE SE 01875 10 Check Kinetic Data Tools
64. Solution High viscosity solution i SA 40 contact time Pe Flow rate Stabilization period Next gt Biacore T200 60 5 Te Kinetics Affinity Samples Samples Sample id MW Da HHH HH 0 01265 a 02530 0 04945 0 08775 0 1955 0 01265 B OO HH 0 01265 a 02530 0 04945 0 08775 0 1955 0 01265 HHHH a a Hun order 9 As entered Increasing concentration Sample id MW Da Z24bkon53ss Concentration nM ug ml Yr cb XJ YJ Next gt 5 1 5 0 0 COO
65. e O Q Quality Control Report Residuals Parameters eo Kinetic constants are within instrument specifications o Kinetic constants appear In be uniquely determined o No significant bulk contributions HI Faund o Check that sensorgrams have sufficient curvature amp Oo oo o Examine the residual plot Pay attention to systematic and non random deviations a 103 107 1 Ms ka 2105 0 5 1 s ka ka Rmox k k
66. sample Show average blank s Include Nex gt Biacore T200 5 89 ES Kinetics Affinity Select Data Create Curves Curve Fc 4 3cor Ligand N A Sample Furosemide Temperature 25 LC ES Lonc Flow Contact Time Diss Time m dn pM l min s s 47 Fc 4 3 corr 30 48 Fe 4 3 corr 30 49 Fe 4 3 corr 30 50 Fe 4 3 corr 30 51 Fe 4 3 corr 30 52 i Fe 4 3 corr i 30 53 Fe 4 3 corr 30 Blank Subtracted Sensorgrams Zoom lock TT eme enne j z Affinity gt ES
67. 133 Run assay step once first Hecurence Repeat assay step within 9 Every 1 cycle Distribute IE accurences evenly Run assay step once first Run assay step once last Cycle Run List 6 2 T Move Up Move Down Startup 1 Startup Thermodynamics 5 times as entered Thermo 1 Sample Thermodynamics 1 time as entered Solvent correction 1 t Conditioning Solent correction 1 time as entered Ewery 15 cycles Control sample 1 t Control sample Thermodynamics 1 time as entered Every 15 cycles o gel rm 30 Startup 1 Solvent correction 1 Control Sample 1 Thermo 1 1 15 Solvent correction 1 Contro
68. 156 U MENSIS E RI REN i i OD RE E 75 76 119 119 V VENDS Sessa MUI MM MU 99 127 143 INGRESE 100 126 143 HOB KGI P RR 127 VW stas ue Lu ud ipuI Du DM DU A RAM DM MI DA UM AL UE 49 W Mas BU er TUDNO i i ii 160 MUEIS RS OIBIEIO eC i i i hG 42 Biacore T200 gr damp ED icio NR RR Rm 17 PF 2A EOE m 53 138 E RR ERR RR ERU NER TUR 54 pw E T appe m 56 PR III TI IIA IS I 25 PUI LT i 23 Pound 23 EE lle ud uU AA E 17 22 A qe III etse duc E M A A M i M E S 182 NULLI c S 25 RR Ko E m 55 zs Er T REMO 1 i ONONO 54 if R E mS NER T T ET 54 gro 24 45 53 54 SM A 26 182 c xc eU N 58 138 EE 39 o s eL M EE 54 OO em 23 Wadi NEC 24 RR 23 Ec 77 78 79 120 121 122 DO UY Zi i 17 c r e IC TE 45 46 52 59 Eau i 46 50 miM EUR A E A TA UNUM MEIN MUSS EE 24 E 6 74 76 117 119 Biacore T200 S UP 7077 017 27 P77 PIFRL 0 62 63 1
69. 2013 GE VSE 15 GE CC 169 0073 Ye n 3 25 1 Intertek ime ada hdd TEL 03 5331 9336 FAX 03 5331 9370 e mail Tech JPG ge com
70. Edit Chip Information Qe te er blew Omn Mea 3 Toms os 4 oet conet Ca mw 4M HE e MWolHvoo o B EEEEEEEEEEE 4 s Z Wee us r EPECEEEEE LEE EE ww gt ow FF lt 4X2 ofocococt Bus iH zh 2131 ee Biacore T200 5 67 5 7 Menubor Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer W nsorgrom pot Baseline AbsResp Binding level Baseline 73 50 RelResp Binding stability Baseline RelResp Binding to reference Binding level Work area Evaluation Explorer Biacore T200 68 5
71. Remove Selection Biacore T200 114 5 ES Kinetics Affinity Fit Kinetics Create Curve Fc 4 3 Ligand antibady Sample antigen Temperature 25 C AddFit Model TBinding 0 w 11Bmdmg 0 w zi Cancel Model _1 1 Binding Add Fil Model 1 1 Binding BM Bivalent Analyte Heterogeneous Analyte Heterogeneous Ligand Two State Reaction Fit Biacore T200 5 115 5 26 RIGET JL B A 1 1 Binding A B AB 1 Bivalent Analyte A B AB AB B AB2 2 2 AB B
72. Minor Gridlines OK RU Adjusted sensorgram I Zoom Lock 350 E 300 4 Tu 250 _ 200 g amp e 150 z 1 8 100 50 0 50 50 0 50 10 150 200 250 300 Time 0 baseline Biacore T200 182 9 9 5 Sensorgram window Copy Graph Biacore Word Pad Pait Word Pad File Edit Wiew Insert Format Help DG E amp d X Bio B Arial v 10
73. Don t Save Save Results From Run s Savein T100manual manual blr My Recent Documents Desktop My Documents 3 My Computer e File name pHscouting My Network Save as type Result file blr Save in File name Sqve Biacore T200 32 3 TER SEA Fil view Run T p I 8x Curve Sensorgram Fc 4 Sample C Lock scale Es o gt m D ke L3 c 0 250 300 Fc Time Window AbsResp SD LASD Slope RelResp Baseline ld Keywords in cycle 2 Value 4 18 0 oo eree 013 003 007 0 0 Yes baseline AssayStep Sample 4 83 0 5 358907 107 52 088 5747 958 9 No binding AssayStepPurpose Sample Buffer Buffer CycleT ype pH Scouting Sample 1 Buffer name 10 mM Acetate 5 Sample 1 Ligand Sample 1 Sample Protein amp 10ug ml Temp 25 Online COMI Temperature 25 00 9C Running immobilization pH scouting Sample compartment temperature current 26 C set 25 C Run time 9 min Estimated run time 18 min Immobilization pH Scouting Standby flow Biacore T200 Evaluation Software
74. Fraction EE 20 20 80 Stabilization period after mix Mix Extra wash after injection with Stabilization period Capture s Enhancement 2 Biacore T200 138 6 Regeneration
75. Biacore T200 50 4 Regeneration command LA Menu bar Commands gt Regeneration Regeneration Vial well position R2B2 Contact time s High viscosity solution Minimum required volume in vralwell For this injection 2 pl 30 60s OK Cancel T Biacore T200 Control Software regeneration check blr ilz File Edit View Commands Run Tools Help HA IE mm Jd B e Cycle 1 m Curve Sensorgram Fc 4 I 2 3 0 B C Lock scale 100 200 500 Fe Time Window AbzHesp SD LRSD Slope HeHesp Baseline Id Keywords in cycle 1 Value 4 1050 5 343 7 011 0 05 0 05 0 0 Yes baseline 1 4 2310 5 3897072 2863 018 1534 22755 No binding 1 4 24650 5 383 857 388 0 21 207 23540 No stability 1 Flow 30 Flow Path 3 4 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 9 min l
76. Control Sample R1A1 R1A12 12 Te 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 3 1 148 Negative control Control sample 40 i 148 Negative control Control sample i 148 Negative control Control sample m e 148 Negative control Control sample i 148 Megative control Control sample 148 Negative control Control sample s M M CX 148 Positive control Control sample F i 148 Positive control Control sample 96 Deep Well Microplate i 148 Positive control Control sample 148 Positive control Control sample 148 Positive control Control sample Us Q Q O O O O O 148 Positive control Control sample T Q O O E OO O 148 Analyte A Sample 10 i 148 Analyte A Sample e C e C e C i 148 Analyte A Sample Q EI Q s 148 Analyte A Sample 3 Q O E O O O i 148 Analyte A Sample O G O O O O C O 148 Analyte A Sample 6 i 148 Analyte A Sample OQ e e Q Q bd bd Q i 148 Analyte A Sample Q OQ Q Q Q 148 Analyte A Sample 00000000 i 148 Analyte A Sample i 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample biem A Back Next gt
77. Curves Include Next gt Biacore T200 84 5 F Kinetics 7 Affinity Select Data Create Curves Curve Multiple Ligand N A Sample 83 Temperature 37 C 15 15 15 15 15 15 15 Blank Subtracted Sensorgrams FF Zoom lock Kinetics gt Ft ES Kinetics Affinity Fit Kinetics Create Curve Multiple Ligand N Sample 89 Temperature 37 C Add Fit Current Fits Model 1 1 Binding v EBEN nes Description 1 994E 5 1 948E 4 9 768E 10 Rmax 1 Cycle 8 Fc 2 1 2 77 nM Rmax 1 Cycle 9 Fc 2 1 0 33 nM i 0 3 300E 10 00 8 330E 9 Rmax 1 Cycle 11 Fc 2 1 0 925 nM i 9 250E 10 Rmax 2 Cycle 7 Fc 3 1 25 nM 2 500E 8 a 2 Cycle 8 Fc 3 1 2 77 nM 2 77UE 9 gt RENT GER ID inc Tis Sg Rmax Fit global Biacore T200 5 85 5 16 Kinetics Summary Y k
78. pH Biacore T200 4 45 4 crude
79. 3 4 Prime 3 _ Biacore T200 7 55 7 1 2 Desorb and Sanitize 1 1 1 Sample compartment 20 A B C D A B C D Biacore Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BIAdesorb solution 2 50 mM Gly NaOH pH 9 5 BlAdisinfectant solution RX 6 ml 80 ml TAR Maintenance Tools Desorb and Sanitize Start l Desorb and Sanitize This procedure removes adsorbed material and disinfects the How system The procedure i divided into five steps Total run time is about ane hour Follewed by a recommended standby time of 3 4 hours NOTE Use the Maintenance Chip tor this procedure The surface on other sensor chips may be damaged by the solutionis used Do not run this procedure below 20 C Do not abort this procedu
80. 40 High viscosity solution Carry over control 40 ul min 30 Evqluation Software Solvent correction 30 ul min 30 InjectAndRecover General Sample Dual Inject Dual Inject 1 2 Generol
81. Next gt Biacore T200 6 147 6 3 T Kinetics7ffinity System Preparations Prime before run C Normalize detector Temperature settings Analysis temperature 25 C Sample compartment temperature 25 tC Cie Gen C Woo ee Prime Normalize Next gt T Method Builder Rack Positions Reagent Rack 2 Sample 1 a8 ive contro 25 Control sample 88 negative control Control sample 88 negative control Control sample 88 negative control Control sample 88 positive control Control sample 88 positive control Control sample 88 positive control Control sample 88 positive control Control sample 88 sample 1 Sample 9E Well Microplate 88 sample 1 Sample 5 88 sample 1 Sample i 88 sample 1 Sample z Q Q Q Q Q Q i 88 sample 1 Sample n O Q C O O O O O 88 sample 1 Sample 10 i 88 sample 1 Sample C o e C C C C 88 sample 1 Sample 00906000 OO O eeoo 88 sample 1 Sample 88 sample 1 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 sample 2 Sample 88 s
82. R RI RI Biacore T200 5 117 Residuals Y B Z 1 lt 2 RU Residuals for a p
83. Sensorgram window _ ect Tools Sensorgram Adjustment E Adjust Sensorgram AX Adjustment Off NA Report Point me CO Injection E vent time 0 Y Adjustment E Report Point response Q Injection Event response 0 Blank Subtraction A C Enable Blank Subtraction X Adjustmentv Report point time 0 baseline istrmernt CQ or Repo Poin ime 0 baseline stabilit OK RU Adjusted sensorgram 1230 E S s E E G v 1225 binding stability ity 1220 w brfdtimgling bindin amp amp binding z sabilit9 stability a settilisl binding stability 1205 1 50 50 100 150 200 250 Time 0 baseline s Biacore T200 176 9 Y Adjustment Report point response 0 C baseline Yr Adjustment ic Report Point response D C3 Injection Event response t OK RU Adjusted sensorgram 30 cu 25 s E binding SIhm
84. Biacore T200 5 101 T Method Builder Main Overview Verification results The method has been verified and can be used to set up a run General Settings Assay Steps Cycle Types Variable Settings a The Method has been verified and can be used to set up arun Setup Run Te Method Builder Detection Detection Elow path 31 Flow path Next gt l Biacore T200 102 5 Startup Sample T Method Builder Variables Variable values for amp ssay Step Sample Sample 1 Sample solution Conc 1 S Conc 2 nm Conc 3 nM E 4 nM Conc 5 nM MW Da 11500 11500 1 063 2 125 4 25 8 5 17 11500 Sample1 Sample Solution
85. 7 Conditioning Startup Solvent correction Calibration Sample Control Sample Undefined Connect to cycle type Cycle types S Recurrence Calibration Control Sample Solvent correction 6 1 Assay step preparations
86. 045 Biacore T200 1 3 1 3 HBS PBS HBS EP 10X 1000 ml BR 1006 69 0 1 M HEPES 1 5 M NaCI 30 mM EDTA 0 5 96 v v Surfactant P 20 ESSUK C 10 0 01 M HEPES 0 15 M NaCI 3 mM EDTA 0 05 96 Surfactant P 20 pH7 4 HBS P 10X 1000 ml BR 1006 71 0 1 M HEPES 1 5 M NaCI 0 5 96 v v Surfactant P 20 i ESSUK C 10 0 01 M HEPES 0 15 M NaCI 0 05 96 Surfactant P 20 pH7 4 HBS N 10X 1000 ml BR 1006 70 0 1 M HEPES 1 5 M NaCI i ESSUK C 10 0 01 M HEPES 0 15 M NaCI pH7 4 PBS 10X 1000 ml BR 1006 72 0 1 M phosphate Buffer 27 mM KCI 1 37 M NaCl i ESSUK C 10 0 01 M phosphate Buffer 2 7 mM KCI 0 137 M NaCl pH7 4 PBS P 10X 1000 ml 28995084 0 1 M phosphate Buffer 27 mM KCI 1 57 M NaCI 0 5 96 v v Surfactant P 20 10 0 01 M phosphate Buffer 2 7 mM KCI 0 137 M NaCl 0 05 96 Surfactant P 20 pH7 4 RRB do1ot TRER 6C ZzEBU C TEN 0 22 um Biacore T200 4 1 1 1 3
87. Biacore T200 164 7 System Check IF the buffer selector shall be tested put tube B C and in water IF all inlet tubes are nat intended ta be used after System Check don t forget ta run Empty Buffer Tubing alter the test A HBS N Buffer Buffer Selector BC D Buffer Selector B CID Next gt 8 System Check Rack Positions Empty Empty vial BR 1004 11 with cap BR 1002 87 min capacity 400p Empty Empty vial BR 1004 11 with cap BR 1002 87 min capacity 200pl Empty Empty vial BR 1004 11 with cap BR 1002 87 min capacity 100pl Empty Empty vial BR 1004 11 with cap BR 1002 87 min capacity 100pl gt BIAtest Solution 1 5 m 795 1 5 ml 4 Merged and Dual injections
88. Biacore T200 Evaluation Software Biacore T200 5 109 5 2 2 Evaluation Biacore T200 Evaluation Software C Documents and SettingsYbiacoreMesktopXT100ManualiCSKASCK antibdy antigen bme Daor Curve Names Overlay e Assay Step Purpose lt Hyerla gt rj Cycle Overlay rj aJ All sensorgrams RU Sensorgram nz E Plot 37100 oom Lock Baseline Sample Binding level Binding stability Binding to reference gt Report Point Table rj Report Point Table F gt Kinetics Affinity AJ antigen mm 1200 1400 Keyword table Tools Keyword Table Edit Chip Information UV 5727 E a a L A 3 aI t
89. Auto Poolingo Yes OK Automatic Positioning Biacore T200 150 6 6 7 7072 088851 Run Stop Run Biacore T200 AN This will stop the run stopRun Run Stopped Finishing current cycle please wait Abort cycle hn Ctrl H Break _ Ctrl Breakl Biacore T200 Evaluation Software Biacore T200
90. Ko Req C X Rmax C Ko Req RU i C M M Biacore T200 56 5 2383 727 21 FEE Ko ED 1 10710 Jo 5 0 72 1 2 n 2 10 0 30 ul min 2424 FC RES 2 2
91. 1 1 Binding Madek 1 1 Binding a BN Brivalent Analyte Heterogeneous Analyte Heterogeneous Ligand Two State Reaction RU Fit Biacore T200 72 5 5 9 B A 1 1 Binding A B AB 1 Bivalent Analyte A B AB AB B AB2 2 2 AB B 2 Heterogeneous Analyte A14 B A1B A2 4 B A2B 1 2 Heterogeneous Ligand A B1 AB1 A B2 AB2 2 Two state Reaction A B AB AB 1
92. 1 2 Empty Buffer Tubing Step 2 Place a bottle containing at least 10 ml 702 ethanol on the left hand plate and insert the Four buffer inlet tubes ABCD 70 10 mD Start 2 3 Biacore T200 7 1199 Empty Buffer Tubing Step 3 Remove the tubes fram the ethanal battle and allow them to hang in the arr This step empties the buffer selector of liquid Set ABCD Start Empty Buffer Tubing The Empty Buffer Tubing procedure is completed Close Close BCID Biacore T200 160 7 7 1 4 Wash Buffer Tubing ABCD
93. Biacore T200 24 3 Ro D 20 RU Rmax x xS Da RU Da S 50 000 Da 1 000 RU 1 20 000 Da Ra J 20 000 x 1 000 50 000 x 1 400 RU 10 000 RU
94. 10 mM HEPES pH 60 80 27 Immobilization pH Scouting Z pH SA 100 ug ml 10 mM Borate 1 M NaCl pH 8 5 NHS pH 8 5 DMSO Instrument handbook Biacore T200 3 27 3 1 1 pH CM5
95. 53 7 1 Toolbar Eject EEF Menu bar Tools Eject Chip Biacore T200 AN This will eject Ehe sensor chip Eject Chip Sensor Chip Maintenance Insert Chip Mew chip Q Reuse chip Mew chip Chip type Maintenance k Chip id DBOB5O09 0135 1387521 Help Dock Chip Cancel Insert Chip Chip type Maintenance Chip id L Dock Chip Dock Standby flow Dock Prime Biacore T200 154 7 7 1 7 1 1 Desorb IFC 1 1
96. Control Sample Control Sample 1 Hecurence Repeat assay step within 9 Every Distribute Themin Startup 3 Run assay step ance fir RARE j Number af replicates Repeat assay step within Thermo 1 Startup 1 Startup Thermodynamics 5 times as entered A Therma 1 Sample Thermodynamics 1 time as entered Control sample 1 Control sample Thermodynamics 1 time as entered Ewery 15 cycles AED Thermo 1 Every lt Distribute d DeL C Run assay step once first Biacore T200 6
97. _ lt Desorb and Sanitize Tools More Tools Service Tools Superclean Biacore T200 158 Y 7 1 3 Empty Buffer Tubing BCD Buffer scouting BCD 20 JY YTRA 70 Tools gt More Tools gt Maintenance Tools Empty Buffer Tubing Start Empty Buffer Tubing This procedure empties all four buffer selector inlet tubes The procedure is divided inta three steps Total run time is about 20 minutes Required solutionis ieinnize water TUZ ethanol Next gt Empty Buffer Tubing Step 1 Place a bottle containing deionized water on the left hand plate and insert the four buffer inlet tubes ABCD Start
98. 4 45 C Concentration unit 5 Buffer settings After run Assay Steps Biacore T200 96 5 T Method Builder Main Startup General Settings Startup Startup 3 times as entered Assay Steps i 2 Sample 5ample Sample 1 time as entered Cycle Types Variable Settings Cycle Run List Assay step properties Base settings Recurrence Name Repeat assay step within Purpose Startup v Every Anis Startup Mi Distribute occurrences evenly Run assay step once first Run assay step once last cycle Assay step preparations 5er of replicates Temperature 3 times Buffer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 Random He sw jJ Sse amp Startup Number of replicates Times 3 i Biacore T200
99. U value 15 25 SE Standard error SE RERE Dxencfisd 9 V2 5x IDRAR SE 01875 10 Check Kinetic Data Tools Modification factor M 1 10 Biacore T200 120 5 k k fgctor M 1 10 Compare to Response
100. slm Ucz S BECd Chiplotno optiona Biacore T200 6 1 Insert Chip Dock Chip Biacore T200 Control Software eo Inserting chip 1 732 Dock Standby flow Standby flow A 7 65 ml 24 3 Dock Insert Chip Cancel Toolbar Eject
101. 151 7 FC Menu bar Tools More Tools Maintenance Tools 2 SOEI A pcd quete s pg Sensor Chip Maintenance E Menu bar Tools More Tools Tools Te Tools S Maintenance Tools Desorb and Sanitize Empty Buffer Tubing Jl Normalize wash Buffer Tubing 2 Test Tools System Check HL Service Tools EX Software Problem Report Flow System Wash 4i Superclean This procedure removes adsorbed material From the Flow system Total run time is about 20 minutes Do nat run this procedure below 20 C NOTE Use the Maintena
102. Kinetics gt 5 8 Remove Selection Blank Subtracted Sensorgrams E Zoom lock Remove Selection Biacore T200 5 71 ES Kinetics Affinity Fit Kinetics Create Curve Fc 2 Ligand antibody 1 ug ml hHH Sample antigen Temperature 25 C Add Fit Z Cancel Model
103. Rmo R Biacore T200 78 5 5 12 F Kinetics 4 Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 1 ug ml pH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v HEHEITT Description Parameters i Delete ka 1 Ms kd 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms RI RU Chi RU U 1 531E 6 0 002610 1 705E 9 7 17808E 77 1 100E 9 2 231E4 8 2 200E 9 2 231E48 4 300E 9 2 231E4 8 8 500E 9 2 231E4 8 1 700E 8 2 231E4 8 Finish Add Fit Model Fit Fs Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Add Fit Model Two State Reaction de 11 Binding DWScriplian L2 Two State Reaction i eee Zo Cey Parameters ka1 1 Ms kd1 1 s ka2 1 s kd2 1 s KD M Rmax RU Conc M tc
104. BIAtest Solution 995 u 7 staqart Biacore T200 7 165 Save hs Save in G Bia Users Q 2 e i MExercsei 4 Methods and Templates My Recent Documents My Documents 4a My Computer e File name KineticsAffinity 1 1 interaction bme hd My Network Save as type Biacore T100 E valuation Files bme v Filename AZJU C Save Buffer_Selector BC D Empty Buffer Tubing l Results System Check System Check Mame ate Instrument Biacore T200 Instrument id 12114 Created By Biacore T200 Control Software Version 1 0 Date 02 5ep 2010 Temperature Z5 File avsCcheck 201009802 Water 2141 2600 to 1400 RU Pags Buffer E 50 tn 50 RUJ Pass Min Max Injection 1 4325 6735 t4000 to 30060 RUJ Pass Injection 2 9603 13198 9000 to 15000 RUJ Pass Mixing Mix 1 47 8 45 0 to 55 0 56 Pass Mix 2 47 8 45 0 to 55 0 96 Pass Difference 0 0 z 5 9 Pass Hefractameter Fc 1 Fc 2 Fc 3 Fc 4 Biatest
105. Next gt l Te Immobilization pH Scouting System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Temperature settings Analysis temperature 25 C Sample compartment temperature 25C Next gt Biacore T200 30 3 LES immobilization pH Scouting Rack Pnsitinns Content Sample 1 Buffer_name 38 Protein 10ug ml Sample 10 mM Acetate 5 5 38 Pratein 10ug ml Sample 10 mM Acetate 5 38 Protein 10ug rml Sample 10 mM Acetate 4 5 38 iPrntein 10ug ml Sample 10 mM Acetate 4 198 50mM NaOH Regeneration u ul Eject Rack Rack tray port Eject Rack Tray Hack Trav Ejected Click DK to return the rack tray to the sample compartment Time In auta cla
106. _ If Then Biacore T200 6 139 Method Variables Evaluation Variables 2 Method Warables E valuation Wariables Method Variables Evaluation Variables Set property as variable Evaluation purpaze Contact time s Dissociation time s Flow rate pl min Extra wash solution Fredefined variables Mame Value type Conc Mumeric hu Numeric User defined variables Value type Method Variables Solution Evaluation Variables
107. kkk 175 9 3 4 ee 176 03 6 Ede Lour m PS B D REM 177 9 3 6 a 0 178 EES m EID EEEE E E E E E A EEN E EE NEEN AEA 180 95 FT iS Dri INTTR 182 SEU ia Bio s RN T NN 184 Biacore T200 1 1 1 1 1 1 1 1 gt gt gt gt Windows biacore DA LED 277 ready JL temperature 1 1 Sample compartment door Sensor chip port Sample compartment
108. 5 97 T Method Builder Main Overview Startup General Settings Delete Startup Startup 3 times as entered Em EE it d cr Sample 5ample Sample 1 time as entered li NEED MEC Variable Settings 4t Move Up a Move Down Cycle Run List Assay step properties Base settings Recurrence Name C Repeat assay step within Purpose Every 1 cycle Connect to Up EON tribut cycle type 2 Distribute Op occurences evenly Run assay step once first Run assay step once last Assay step preparations Number of replicates Temperature g times Buffer j As entered 1 2 3 1 2 3 Order 1 1 22 331 O Random Sample Number of replicates Times Cycle Types Biacore T200 98 5 T Method Builder Lose 2 Varieti Seng A JSO Description of selected cycle type This cycle is used in sample steps and if used control sample steps Contains injection of sample and regeneration The sample is of the type single cycle k
109. 9 4 Sensorgram window caption Undo Cut Scale Copy Graph Export UTE5 Gridlines Legend 27 OZE Scale Scale Y Scale Auta Auto Logarithmic Lagarithmic Auto te Z Min Max A Scale Y Scale Auto amp uto Logarithmic Lagarithmic OK Biacore T200 9 181 Legend Legend Position O Hidden O Lef Q Top 5 Right C2 Bottom Right Gld Hidden OK Gridlines Gridlines m AMS Major Gridlines C Minor Gridlines Y Axis Major Gridlines Minor Gridlines Mojor Gridlines
110. T Method Builder Main 2 Assay steps General settings Concentration unit nM General Settings Startup Data collection rate 10Hz kineti d Sample compartment temperature 25 C Startup LMW kinetics 3 times as entered Dac m Assay Steps A Sample Cycle Types Sample LMW kinetics 1 time as entered Settings for assay step Startup Temperature 25 C Variable Settings Solvent correction Buffer t Solvent correction Solvent correction 1 time as entered Before after every Verification Control sample m t Control sample LMW kinetics 1 time as entered Before after ewery Settings for cycle type LMW kinetics HH Sample 1 varies by cycle 60s 600s HH Carry over control 1 SetupRun Report points Expand lI Collapse All General settings Biacore T200 128 6 T Method Builder Main Overview Data collection rate Sample compartment temperature AssawSteps Cycle Types A settings Hz Dul y C Vary with analysis temperature Variable Settings Concentration unit vewcaon
111. 5 65 Saye Results From Run s Savein T100manual Immobilization of antibody blr Immobilization of Prteina blr manual blr s pHscouting blr regeneration check blr regeneration check wizard blr surface parformance wizard blr Thermodynamics antibody vs antigen blr File name Kinetics antigen antibody D My Network Save as type Result file blr Save in File name Sqve l Standby flow l Biacore T200 Evaluation Software 5 5 7072 LORSE Run Stop Run Biacore T200 AN This will stop Ehe run EIN Stop Run Run Stopped Finishing current cycle please wait Abort cycle hn Ct Break _
112. nag Parameter Setting OK Fit Biacore T200 5 123 5 30 Current Fits Current Fita 1 1 1 Binding Description Two State Reaction Delete Biacore 100 Evaluation 4 J Are vou sure you want to delete this Fit OK Curent Fits 1 Two State Reaction Description Biacore T200 124 6 o
113. 3 5 NHS LEM immobitzation Results Chip CMS Response Response Ew cell Procedure Method Loa nd Bound RU Final Rtf 3 Target Reached Target level mne antibody 160ugfml pHs Ha Proconcentration binding is tex Fari Preconcentration binding is too fast pH Preconcentration binding is too slow
114. HBS N Buffer 150 ml 10X Buffer Series S Sensor Chip CM5 BlAtest solution 1 5m CM5 Dock HBS N Prime Tools gt More Tools gt Test Tools gt System Check Start System Check Select test s to run This procedure should be run at 25 C with a new Sensor Chip CMS and with HB5 M as running buffer Choose Clase if vau need to change the sensor chip reset the temperature ar change running buffer IF any injection is delayed adjustments can be made by the software The required test is Injections and refractometer Reagent pumps and blank injection Injections and Refractometer Mix Moise Merged and Dual injections optional Buffer selector optional Tests if the peristaltic pump iz in order and that a sample injection with buffer from the reagent supply black is all right System Check 4 Next gt Merged and Dual injections Buffer Selector
115. Kinetics Affinity Fit Kinetics Create Curve Fc g Ligand antibody 1Uug ml nHH Sample antigen Temperature 25 LC Add Fit Current Fits Model 1 1 Binding hr 1 1 Bindini Description Guality Control Report Residuals Parameters Biacore T200 5 73 RGI2e ZZ 2 9 1 1 Binding FED _ 5 10 Quality Control 5 O08 Quality Control Report Residuals Parameters eo Kinetic constants are within instrument specifications o Kinetic constants appear In be uniquely determined o No significant bulk contributions HI Faund o Check that sensorgrams have sufficient curvature amp OE SB o Examine the residual plot Pay attention to systematic
116. pH Control Software Sensorgram Plot aul Bar Chart AJ Kinetics Affinity Concentration Analysis 7 Thermodynamics Evaluation Explorer Dj E Adjusted sensnrgram DE a Curve Name Fc 4 P 4 Assay Step Purpose Sample Cycle Overlay P ensorgram j Adjusted sensorgram RU Adjusted sensorgram Report Point T able 3000 Zoom Lock m Report Point T able k PE 10 mM Acetate 4 10 mM Acetate 4 5 10 mM Acetate 5 10 mM Acetate 5 5 Response 0 baseline 50 100 Time 0 baseline Biacore T200 3 33 3 1 Immobilization pH Scouting Adjusted sensorgram Zoom Lock 10 mM Acetate 4 10 mM Acetate 4 5 10 mM Acetate 5 1 mh Acetate 5 5 H 8 i 3000 50 50 100 150 200 250 pH pH4 pH NHS NHS pH8
117. BSA _ 30 Biacore Maintenance Kit type 2 BlAdesorb solution 1 0 5 96 SDS BlAdesorb solution 2 50 mM Gly NaOH pH 9 5 Tools gt More Tools gt Maintenance Tools Wash Buffer Tubing Start Uv2Us3 Wash Buffer Tubing Select tubes ta wash Al four tubes Next gt l Wash Butfer Tubing This procedure removes adsorbed material from the Flow system The procedure is divided into three steps Total run time is about 30 minutes HOTE Use the Maintenance Chip for this procedure The surface on ether sensor chips may be damaged by the solutions used Do not run this procedure below 20 C Do not abort this procedure after it is started Next gt Biacore T200 7 161 Wash Buffer Tubing Step 1 Place 20 ml Bl amp desoerb Solution 1 on the left hand tray and insert tube a BIAdesorb Solution 1
118. CM NHS N NHS CON COO COO CONHCH CH OH ONKS BR 1000 50 EDC N ethyl N C3 dimethylaminopropyl carbodiimide hydrochloride NHS N hydroxysuccinimide 1 M ethanolamine hydrochloride pH 8 5 EDC NHS 10 ml 400 mM EDC 100 mM NHS 200 ul 7 mm 20 C 2 DA 1
119. k Affinity Y Kp X Biacore T200 Kinetics Summary Append File File Append File Tools Kinetics Summary Biacore T200 Kinetics Summary Thumbnails ER Biacore T200 Kinetics Summary 1 bme m Display Settings Eko Small Thumbnails Standard Thumbnails Extended Thumbnails File gt Save a
120. stability 1 3 10 y 1 Inject Ready 20 yl e e9 baseline 1 RU o EO RU RelResp 0 0 binding_1 lt stability 1 RelResp baseline_1 RU 2 baseline_2 binding 2 stability_2 RelResp boseline_2 RU Toolbar Reference line Menu bar View Reference Line l Biacore T200 2 21 LE Biacore T200 Control Software manual blr ilz File Edit View Commands Run Tools Help rd p gl pr y e Cycle 1 a Curve Sensorgram Fc 1 GxEI22zZIs3IoIB JEN Sj C Lock scale ttl Y 409 60000 x binding 1 amp 45000 Wash Starf stability 1 Inject Read 50 100 150 350 400 Fc Time Window AbsResp SD LASD Slope M HRelResp Baseline Id Keywords incycle 1 Value 1 210 0 5 358350 002 2003 0 00 0 0 Yes baseline 1 1 275 0 5 580110 2 00 014 1 06 221150 No binding 1 1 290 0 5 3583852 0 54 0 60 004
121. 3 267 7 4 266 9 Contact time 5 s Flow rate 30 l min Stabili 5 267 6 6 267 9 7 269 2 9 2682 Sample Relative response stability T e e o 15 Cycle number 20 Biacore T200 5 53 5 BiacoreT200 6 Kinetics Affinity 54 Concentration Analysis Binding Analysis
122. 4C 200 ul 1 1 BSA 10 mM 10 mM HEPES ER 10 mM Borate 1 M NaCI pH 8 5 Biacore T200 26 3 0 5 2 pH 5 200 ug ml e 10 mM BEBSZ I UD AED pH 4 0 5 5 pH 3 5
123. Q amp A www gelifesciences co jp gt FAQ Q amp A Q TEL 03 5331 9336 O MicroCoal O O Q FAX 03 5331 9370 e mail Tech JP ge com Q amp A Web TEL 03 5331 9315 FAX 03 5331 9349 TEL 05 5551 9550 FAX 05 5551 9524 WWW Qelifesciences co p e mail Web
124. 1 nM 1 uM 5 5 Ks 1879 5 Ze AA 0 30 ul min 2424 Fu C REESE 2 2 5 10 10 30
125. Menu 73 5 Automatic Positioning T Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Si i First Sort By Control sample 7 Cyan i Column Bottom left Sample Small Ascending Sample B CarkElue Column Bottom left Sample Small Ascending Startup II Crimson Column Bottom left Sample Small Ascending Wash E vellow Column Bottom left Reagent Large Ascending Solvent correction buffer amp Il Blue Column Bottom left Reagent Small Content Ascending gt Pooling Auto Poolingo_ Yes OK Automatic Positioning Biacore T200 106 5 l Eject Ra
126. Methods and Templates 2 Open Browse Open Te Immobilization pH Scouting Setup Detection Elow path Buffers Buffer Name 10 mM Acetate 10 mM Acetate 10 mM Acetate 10 mM Acetate Flow path SEX CJLTGESR UJ KARRE Next Biacore T200 3 29 Te immobilization pH Scouting Injection Parameters E Ligand Solution Protein 10ug rl Contact time s Flow rate ulmin Surface regeneration This surface wash will be run ance at the end af each cucle Solution ml MHaQH Ligand Solution contact time Cs 60 s Flow rate umin 10ul min Surface regeneration Solution 50 mM NqOH
127. Conc nM Conc 1 Conc 5 MW Do 2 _ Next gt Biacore T200 5 103 T Method Builder Cycle run list buffer buffer buffer antigen antigen antigen Next gt Te Method Builder System Preparations Frime before run Normalize detector Es Es Prime Normalize Temperature settings Analysis temperature 25C Sample compartment temperature 25C Next gt Biacore T200 104 5 T Method Builder Rack Positions Sample and Reagent Rack Sample 1 Pe 118 antigen Sample 0 11500 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample coo oo oo Ogm 118 antigen Sample e 118 antigen Sample 118 antigen
128. Eject Rack Tray Rack Positions Next To Kinetics Affinity Prepare Run Protocol Prepare Run Protocol e 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 yials should be sealed with rubber caps and microplate with adhesive foil e Place the buffer s on the left hand tray and insert the correct tubing s see below Note Standby after run will use buffer A ke sure there is fresh water in the water bottle on the right hand tray f the run res le e fnecessar y empty the waste bottle before start o Estimated run time 4h 29 min excluding conditional statements temperature changes and standby flow Estimated buffer consumption ARUXE BS ODERASBUGHORERASIRCORREBSRG ERI LLL 7 RSDROSJ zumnctf dg Start Sl amp Save as Methods and Templates 2 Bia Users FLY Don t Save Save in File nime Save
129. Flow cells per cycle 1 Flow cell 1 o Immobilize flow cell 1 Flow cell 2 Fi E Immobilize flow cell 2 Flow cell 3 Immobilize flow cell 3 Flow cell 4 O sl Immobilize flow cell 4 Chip type CM5 Flow cell 4 EI mml dios eel d Methad 9 Aim for immobilized level Ligand Dilute ligand 3 Specify contact time and flow rate Target level RU wash solution 50 mh NaOH C3 Blank immobilization Flow cell Aim for immobilized level Method Amine Ligand Target level RU Wash solution 50 mM NaOH Next gt l 37 40 l Biacore T200 3 43 Te Biacore T200 Control Software immobilization of antibody blr mf iz Fie Edit view Run Tools Help om X E aa lc 7 Spl Cyde 1 Curve Sensorgram Fc 4 antibody C Lock scale 4 TS siii 4 500 1000 2000 Time Window AbsResp SD LASD Slope Rel
130. M 12 RU 60 50 40 30 an 10 1e 22 6 3e B ie B 5s B e Te e Je 1 5 Concentration hi Report Parameters LB 9 Rman RU offset Ru me us a b41E 7 0 0545 2 05 0 02560 4s B 5e 6 Concentration Report Parameters KD 9 Rman RU offset Ru ch RU o DIE 5 D 524 0 93490 Biacore T200 5 93 5 2 54 54 Kp ka ko 55 _ Kp 1 10 10 GOREESBBH C 5
131. 13 1 11 Reagent rack EI AgI ABC 123 A1 A2 A BC D EF G Biacore T200 14 2 2 3 Toolbar t Biacore T200 Control Software File View Run Jac Help 7 3 370639 EIE Manual run _ 4 Application wizards J4S97AIlWUVa 5 X SEFeEAJZUCEGQGUS42 HFt FcC3S UdFr5xol aBirttbEEixs EFRA U C947 z22A2cox SRL CCODERFHZ 4 U FN pH RADIF 4 278 C XERRERT EORR amp Bb U 78 cy Methods
132. 2 Heterogeneous Analyte A14 B A1B A2 4 B A2B 1 2 Heterogeneous Ligand A B1 AB1 A B2 AB2 2 Two state Reaction A B AB AB 1 antigen m fx Curve Fc 4 3 Ligand antibody Sample antigen Temperature 25 C Fie 1 1 1 Binding E I i B nto A h M H Quality Control Report Residuals Parameters Biacore T200 116 5 1 1 binding Quality Control 5 27 Quality Control 5
133. Biacare T200 Evaluation s J Are vou sure you want to delete Ehis Fit OK Curent Fits 1 Two State Reaction Description Biacore T200 5 81 5 14 Batch mode Select Evaluation mode C Batch mode Batch mode FS Kinetics Affinity Select Curvec Create Select evaluation mag Single ode Batch mode Evaluation settings Evaluation purpose Kinetics Model E 1 1 Binding Samples Curve type ReferenceSubtracted v Include Curve Ligand Sample v Fc 2 1 antibody 10ug ml pH5 antigeni v Fc 2 1 antibody 10ug ml pH5 antigen2 v Fc 2 1 antibody 10ug ml pH5 antigen3 Check All Uncheck All Ens Less Samples Evqluation purpose Model Kinetics Affinity Calculating Results Samples Fitting sample 1 of 3 antibody 1Hunrml pH5
134. Biacore T200 6 143 Variable Settings Te Method Builder Main Assay steps Define variable handling for each amp ssay Step General Settings Sample O Define all values at run time Solvent correction Control sample Define all values in method 9 Define some values in method and others at run time Variable Settings __ Verification Enter values for the variables in this assay step Setup Run ue s ssess 3 Define all values at run time Define all values in method lFERUIESXVvE amp SRSEICTLZUL Define some values in method and others at run time
135. Cel Eject Chip Insert Chip Toolbar Insert Ea Biacore T200 1 7 fig 1 6 Reuse Chip Insert Chip New chip 9 Reuse chip Reuse chip Reuze CM5 Id 080507 1029 12114 v Chip type CM5 Chip id 080507 1029 12114 Chip lot na Reuse id Details Chip Properties Chip id Chip lot no First use date 0s050r 1029 12114 DE IFC type IFC1 05 Immobilization Final Response Foment Men RU E Ez i2008 antibody CilBia LlsersiTi ManualcsKlimmoabilization of antibody blr Close Close id id
136. EDC 89 ul 7 mm NHS 89 ul 7 mm NHS EDC 7 mm Ethanolamine 129 ul 7 mm Ligand 98 ul 7 mm Eject Rack Rack tray port l OK Eject Rack Tray Rack Positions Next gt Te immobilization pH Scouting Prepare Run Protocol Tahoma ll 10 B 7 U Prepare Run Protocol Make sure the correct sensor chip is docked 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 fail Place the bufferis an the left hand tray and insert the correct tubingis see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 18 min excluding conditional statements temperature changes and standby Il Estimated buffer consump
137. Toolbar qp Kinetics Affinity 4 21 y pg A Surface bound amp Kinetics Affinity Select Curves Create Select evaluation made Single mode Batch mode Curves Curve Ligand antibody 10ug ml pv Sample antigen Temperature 5 vw CUCIEN Conc Flow Contact Time Diss Time iniia nM ul min s s 3l 1 1 v v La L4 v v v L4 Zoom lock Shom concentration series Show blank s Show average blank s Select Evaluation mode 1 Single mode Batch mode cd Bath mode 5 14 Sqmple Show average blank s
138. M v SeupRun After run 5 Specify analysis temperature after run General settings 6 Data Collection rate 1Hz 10Hz 10Hz 1Hz Detection 3 Single Dual Mult 725 Single 1 2 3 4 Dual L y 3 44 el 4 5 Multi 12 544 2 1 4 4 2 1 5 1 4 1 Sample compartment temperature 4 45 C 10 DMSO Biacore T200 6 129 Vary with analysis temperature zr BA Analysis temperature Concentration unit Buffer settings
139. 8 5nnE 9 2231E 8 0 1167 1 7DDE 8 2 231E B 0 1062 H ka 1 Ms ka 1 5 FERE TER ES XE EL Ko M FERE AE AN Rmax RU RI RU bulk effect Chi RU U value U XU 3 14 Binding Finish Evaluation Explorer Biacore T200 76 5 gt Sensargram 4 All sensorgrams Plat a Baseline Sample a Binding level Binding stability g Binding to reference Report Point Table Hrepen Fant T able Kinetics Affinity PJ antigen Toolbar AJ Kinetics Affinity HE 5 11
140. EEBOIBNICIAU x o o iB JE OANA HE SP e c6REOXS e5X33J2 QU e o lE Pe
141. Toolbar Run Wizard 7 Menu bar Run gt Wizard m Open New Wizard Template CI Surface Preparation Immobilization pH Scouting 2 GI Assay Development Regeneration Scouting w Buffer Scouting A Surface Performance CI Control Experiments Kinetics Linked Reactions Kinetics Mass Transfer E Assay Look in a3 Methods And Templates Name Kinetics Affinity Binding Analysis Concentration Analysis Thermodynamics CI Immunogenicity Immunogenicity Screening 1 Immunogenicity Confirmation Immunogenicity Isotyping Surface Preparation Immobilization New Methods and Templates 2 Open Browse Open Biacore T200 42 3 8 Immobilization Immobilization Setup Chip type CM5
142. un 13392 Iniect Stat R2B4 Report point Fc Time Window AbsHesp SD LASD Slope RelResp Baseline d Kewgl 1753 2 Inject Ready 70 pl 4 d 359025 007 007 0 02 Q0 Yes Baseline Chip 1 723 Wash Stat 360287 010 004 005 1261 No EDC NHS Contaci 18029 Wash Ready 374125 088 003 047 15099 No Ligand FlowRa 1918 0 Temperature 25 00 C 344165 040 004 021 15139 No Immobilized Ligand OU Method Amine Procedure TimeandFlaw 32000 0 200 400 600 800 1000 1200 1400 4 Fao table 4 18130 mn on Keyword table Statu S ba r on ine 1 Temperature 25 00 9C Running method Sample c mpartment tempera urrent 40 C set 40 C Run time 6 h 55 min Estimated run time 15 h 7 min Online COMI Temperature 25 00 C Running method Sample compartment temperature current 40 C set 40 C Run time 6 h 55 min Estimated run time 15 h 7 min Menu bar Biacore T200 Toolbar Sensorgram window Report point table Event log Status bar
143. wi 1 g 8 t t ins ind iC r 74 2 EET S i t ss ee es La ee o es se ssa ss ss zes med res 2 ERTER Biacore T200 110 5 5 24 Menubar Toolbar Evaluation Explorer gt Work area Menubar Toolbar Evaluation Explorer W nsorgrom piot Baseline Binding level Binding stability e m Report Point Table Binding to reference Work area Biacore T200 Sensorgram window AbsResp Baseline 73 50 RelResp Bqseline RelResp Binding level Evaluation Explorer 5 111 Toolbar qp Kinetics Affinity 4 51 y pg A Surface bound amp Kinetics Affinity Select Curves Create Select evaluation mode Single mode Batch mode
144. 1 2 Next gt Recommended settings are nat followed Sample serie sample The sample series should contain at least one sample with zero D concentration The sample series should consist of at least five B different concentratans The sample series should contain at least one non zero concentration that is to be run at least two 2 times Ilgnore Biacore T200 5 61 BXE 5 2 Excel Excel Excel txt LM Kinetics ffinity System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C P
145. 1e MES OE TU FSRXE NS RHAKGGERIEJJ AES NFEDRSUM HFE FC3S Manual run Biacore T200 2 15 2 1 Toolbar Start Manual run Ep Menu bar Run Manual run Te Manual Run Flow g Flow rate ER min Flow path Reference Detection in flow cell sr 1 2 3 4 subtraction e E Flow path 1 Q E Flow path 1 2 Q 3 Flow path 2 Q EJ Flow path 3 4 O a Flow path 3 Flow path 1 2 34 C a Flow path 4 Flow rate WRIA 1 100 ul min Flow path Rack _ Start _ Start 2 1 ul min s ul 28 pl Pe
146. 2 s2L Bto20 t vs x Reference Active _ ReferenceSubtracted Ctrl Lo All sensorgrams Curve Names Overlay v 4 Assay Step Purpose verlay rj Cycle lt Dwerlawy P RU Sensorgram 38000 Zoom Lock 37500 37000 36500 35000 35500 35000 34500 34000 0 Biacore T200 9 173 E Cycle Overlay 7 o U qal a 50 v 2L LE Lyclez Assay Step Purpose Sample Name JI EE 1 Startup Buffer E T 4 Sad 0 m BER a 3 Startup Buffer Yes Jg Sample Betazmicro 32 11800 Yes Bioampe Betazmicra 16 11800 Yes M 7 Sample Betazmicro E 8 11800 Ctrl
147. 70 On rate ka log 100 50 0 50 100 150 200 250 300 350 Model 1 1 Binding ka 1 Ms 1 531E 5 100000 BEES kd 1 s 0 002610 Off rate kd log ka 1 Ms kd 1 s KD M Rmax RU Chi RU2 Ligand Model Evaluation File 5 60e4 05 2 04e 03 3 65e 09 108 7 0 112 antibody 10ug ml pH5 1 1 Binding 1 1 53e406 2 61e 03 1 70e 09 105 3 0 1 antibody 10ug ml pH5 1 1 Binding Fc 2 1 1 2 19e405 1 63e 03 7 44e 09 114 3 0 0501 antibody 10ug ml pH5 1 1 Binding 1 Dox m Table Columns Selected Evaluation Ligand a Sample antigenl 0 2e 9 4e Be Se 9 1e 8 1 4e 8 1 8e 8 Model Steady State Affinity KD M 1 359E 8 Sample Sample KD M Rmax RU Control Rmax RU 100 Da Chi RU Model antigen3 3 59e 08 198 3 NJA 0 0462 antibody 10ug ml pH5 Steady State Affinity antigen3 3 59e 08 198 3 N A 0 0462 antibody 10ug ml pH5 Steady State Affinity antigen 1 37e 08 165 1 NJA 0 517 antibody 10ug ml pH5 Steady State Affinity gt Steady State KD Plot Kovs Sampe Affinity Kbp
148. Before after every Control sample t Control sample LMW kinetics 1 time as entered Before after every Settings for cycle type LMW kinetics HH Sample 1 varies by cycle 60s 600s HH Carry over control 1 Report points Expand lI Collapse All Overview 6 2 Overview General Settings Assay Steps Cycle Types DNN V arable Settings Verification General Settings Verification 5 Overview General Settings Biacore T200 6 127 Assay Steps Cycle Types Variable Settings FERA DIED RE Verification Overview
149. Curves Curve gend antibody E Samne law Cycles Conc Flow Contact Time Diss Time dii nM ul min s s L 4 30 Ti 5haw concentration series Show blank s Show average blank s Sample GRID Show average blank s include Biacore T200 112 5 ES Kinetics Affinity Select Curves Create Select evaluation made Single mode C3 Batch made Curves Curve Fe 4 3 wl Ligand antibody v Sample antigen bd Temperature 25 Cycles Conc Flow Contact Time Diss Time SENS nM ul min s s 4 30 o o D D D D Zoom lack
150. Flow ul min 1 554E 5 0 002932 5 060E 4 1 866E 6 5 790E 12 106 0 7 145E4 7 1 100E 9 2 200E 9 4 300E 9 8 500E 9 1 700E 8 se Le E Current Fits Current Fits Finish Biacore T200 F Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 10ug mlpH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v 1 1 1 Binding Description Delete 5 79 ka 1 Ms kd 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms RI RU Chi RU2 U 1 531E 6 0 002610 1 705E 9 105 3 7 178Ec 7 1 100E 9 2 231E 8 0 05105 2 200E 9 2 231E 8 0 2011 4 300E 9 2 231E 8 0 1965 8 500E 9 2 231E 8 0 1167 1 700E 8 2 231E 8 0 1062 Rmax R Add Fit Parameters Parameter Settings Two State Reaction Fit Initial value Fit global le5 Default Fit global 1e 2 Default Fit
151. Kinetics Affinity Select Affinity Data Create Curve Fc 4 3cor Ligand N A Sample Furosemide Temperature 25 C Seting Calculate response at position 4 seconds before injection stop with window 5 seconds 58 5 Concentration Fo Wh amend Biacore T200 90 5 RU Req RU Req Next gt Y Kinetics Affinity Fit Affinity Create Curve Fc 4 3cor Ligand N Sample Furosemide Temperature 25 C Add Fit Model Steady State Affinity aa Parameters 5e 6 Concentration X MV Y Req RU Model Ft Steady State Affinity 5 18 3 Steady State Affinity 1 1 Binding Rnmex Fitting Req Kec offset Steady State Affinity Constant Rmax 1 1 Binding Rmax
152. Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 118 antigen Sample 154 buffer Startup D 5 20 Menu Export Positions D Menu gt Simple Position Import Biacore T200 5 105 5 21
153. Show concentration series Show blank s Shaw average blank s uplg Amas Next gt Kinetics Affinity Select Data Create Cumes Curve Fc 24 3 Ligand antibody Sample antigen Temperature 25 L 3 Blank Subtracted Sensorgrams C Zoom lock z Biacore T200 5 113 0 Kinetics gt 5 25 Remove Selection Blank Subtracted Sensorgrams E Zoom lock
154. Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 2 min Cancel Inject Biacore T200 2 19 Eject rack tray CL Menu bar Commands Eject Rack Eject Rack Tray Hack Trav Ejected Click DK to return the rack tray to the sample compartment Time In auta clase 50 Ok l Inject Vial well position R2 B1 Contact time 3 Minimum required volume in viral well For this injectien 58 ul Inject command OK Biacore T200 Control Software manual blr ilz File Edit View Commands Run Tools Help id E En B y e Cycle 1 m Curve Sensorgram Fc 1 exSSI3IGIB C Lock scale 13 42 ls M B KP Inject R2B1 60 50 100 AbsResp SD LASD Slope HalHesn Baseline Id Keywords in cycle 1 Value 358960 0 02 0 03 0 00 D
155. and non random deviations a 103 107 1 Ms ka 2105 0 5 1 s ko ka Rmax I k paret RI RI RI Biacore T200 74 5
156. e 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 30 3 63E 09 36 M 4 MINFile Properties Baseline Sample Binding level Binding stability Binding to reference Beta2micro Chi RUJ 0 136664 0 178485 0 31193 0 113137 0 242235 0 992655 0 212286 m Biacore T200 184 9 9 6 Fille gt Save As Save As Cy Methods and Templates My Recent Documents 2 Desktop Mn Documents L Mn Computer File name Einetics amp ffinitu 1 1 interaction bme v My Network Save as type Biacnrs T100 Evaluation Files bme wv Save in C Bia Users File name Save fig 9 2 Biacore T200 Control ha Biacore T200 Evaluation Biacore T200 A P Io SDORNSOIIE Sedes cttas etu uUa epe DM DOR EU n EDU A 21 Aterro i 95 129 ATON Oea ENE hifi 35 42 PPan TET aU E aE E
157. global le Default Fit global le Default Fit global hax Default Fit global 188 Default Fit local Ya DeFault Rmo Ras Fit 02407 amp 2U v 2L Fit local 0 RI RI Fit Constant Initial value 0 Parameter Setting OK Fit Biacore T200 80 5 E 5 13 Current Fits Current Fita 1 1 1 Binding Description Two State Reaction Delete
158. injection window Rack tray port 2LLVZBEVARO D Biacore T200 2 _ 1 1 1 2 A CO a A B C D A BCD 500 ml
159. ka Sus Ko A B AB Ko ka ka KA Ka ka Biacore T200 5 55 Kp ka k R f ka ka Rmax C k kg Rea Reqvs C C Req Ka
160. solution z2309 zzz59 z2207 zzzu4 21400 to 23600 RU Pass variation laz B n RL Pass Baseline lewel 36001 35893 35843 36176 variation 333 3000 RUJ Pass Injections v PASS FAIL FAIL N LI Biacore T200 166 8 8 7 7 8 1 Standby flow A 65 ml 24 7 St
161. tubes Flacg water on the right hand tray and insert the water inlet tube A B C D Start 4 5 Desorb and 5anitize Step 5 Place tube A in a HEPES ar THIS buffer Recommended concentration 10 50 mmol l Let tubes B C and D hang in the air A A 10 50 mM HEPES Tris B C D Start Hesnrh and 5anitize The Desorb and 5anitize procedure is completed Allow the zustem ta run in standby mode for at least 3 4 hours before performing a run 5 3 standby flow 3 4 Prime 3 l Close Prime
162. 0 03 10 2 No stability 4 Sample 1 Conc H5 17 328 8 5434 0414 013 0102 143 No binding 5 Sample 1 Ligand antibody 343 8 5420 012 0 12 0 01 123 No stabilitu 5 Sample 1 MW 11500 Online COM1 Temperature 25 00 9C Sensor chip CMS cn ka cn cn ac cn c on co oi on Sample compartment temperature current 25 C set 25 C Running standby remaining time 4 0 days l Standby flow i Biqcore T200 Evaluation Software Biacore T200 108 5 fig 5 22 Run Stop Run Biacore T200 AN This will stop the run stopRun Run Stopped Finishing current cycle please wait Abort cycle hn Ctrl H Break _ Ctrl Breakl
163. 0 8 No stability 1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run Lime 7 min RE Toolbar Add Report point E Menu bor O Edit gt Report point Add Report Point Report Paint e Time 258 0 s Window i Baseline Add ta all curves in this cycle Id 0 Baseline OK Biacore T200 22 2 2 1 3 RIED S End Manual run Cp Menu bar Commands gt End Run Standby flow 2 2
164. 04 105 147 149 prO ME bh 61 66 87 109 L ILO E IRR T TRES 162 zio sia cA c i 163 LM Nd EE i e T i if 93 zL rlp bl dm vae e A 138 arsUpnld INEA 15 xS E Peg e CUM FOTO 45 53 54 136 gd pL 2 24 E E PEN NEC 166 ir E Mr EU NR NUR ERREUR A 73 116 Le sm 40 169 172 poH pU JOBE E IS iue santettieidulsdeied tetigere inedite utendo ementi 7 BODE i 5 CJI LRS RET 167 eo 12 22 DD i fi i i 58 99 RE San 182 KN RA c i 182 ESED E bi fh 166 24 pr n 178 B Wr SN 12 uc co 77 68 USUS taz S NER T T TA 45 RD ER hh 71 72 114 DE 55 Biacore T200 rco 76 119 NEUE OU Ssb 184 p Rond CHEERS 117 Wa 63 105 149 sab MIL 27 Sr RE 7 RR E RRRRRRRRRRRRE 87 c LAFZI AT PUST A S TET 24 73 116 rm t 15 UD UU RNN 53 54 Da rU fil 143 SL Nr 14 124
165. 1 Binding Description Delete ka 1 Ms kd 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms RI RU Chi RU2 U 1 531E 5 0 002610 1 705E 9 105 3 7 178Ec 7 1 100E 9 2 231E 8 0 05105 2 200E 9 2 231E 8 0 2011 4 300E 9 2 231E 8 0 1965 8 500E 9 2 231E 8 0 1167 1 700E 8 2 231E 8 0 1062 fh V2 2 9 Rmax R Add Fit Parameters Parameter Settings Two State Reaction Fit Initial value Fit global le5 Default Fit global 1e 2 Default Fit global le Default Fit global le Default Fit global hax Default Fit global 188 Default Fit local Ya DeFault Rmo R Fit 222407 amp 2U vL Fit local 0 RI RI Fit Constant Initial value 0
166. 126 L Lor d D NR ERREUR 151 153 26 46 DOT Y eT 45 73 75 91 116 118 ION ecce Duce MM RI MM M EM M D DE 132 124 138 141 5 DN NE E EE TEES 11 12 13 PAS E ND EN NE AANEEN A A TE 17 DIA E T OLS 9 or E UE T EOD E T EA A EE AO 3 ERE EE NET 25 26 27 28 LL IR i TN 23 TRA 35 45 DD viv do ET 20 21 50 51 Pel vac pas LE 51 hi 17 MEOT E K 17 Prnt 4 hG 20 22 50 67 110 138 140 Biacore T200 20 20 Biacore T200 XD WSJAZSSUAS sSLL CES rine nad NN Cle b Ux TOSS vm I i E IH IC fees D 9
167. 20 m Stqart 1 2 Wash Buffer Tubing Step 2 Wipe the tube with a moist tissue Place 20 ml Bl desarb Solution 2 an the left hand tray and inzert the tube stat BlAdesorb Solution 2 20 ml Start l 2 3 Wash Buffer Tubing Step 3 Wipe the tube with a moist tissue Place buffer ar water on the left hand tray and insert the tube Set Start l 3 Wash Buffer Tubing The wash Buffer Tubing procedure is completed Close Biacore T200 162 7 7 2 7 2 1 Normalize Biacore Maintenance Kit type 2 BlAnormalizing solut
168. 31E 8 1 700E 8 2 231E 8 Finish Add Fit Model Fit Fs Kinetics Affinity Fit Kinetics Create Curve Fc 2 1 Add Fit Model Two State Reaction Parameters Tools w Parameters ka1 1 Ms kd1 1 s ka2 1 s kd2 1 s KD M Rmax RU Conc M tc Flow ul min kt RU Ms 1 554E 5 0 002932 5 060E 4 1 866E 6 5 790E 12 106 0 7 145E 7 1 100E 9 i 2 220E 8 2 200E 9 2 220E 8 4 300E 9 i 2 220E 8 8 500E 9 i 2 220E 8 1 700E 8 i 2 220E 8 gt Current Fits Current Fits Finish Biacore T200 122 5 RENT V2 54 9 COBENTRR XE SE TEOD Z5 B F Kinetics 7 Affinity Fit Kinetics Create Curve Fc 2 1 Ligand antibody 10ug mlpH5 Sample antigeni Temperature 25 C Add Fit Current Fits Model 1 1 Binding v 1 1
169. 5 pH pH pH5 Immobilization pH Scouting RU 100 ug ml Immobilization pH Scouting Biacore T200 34 3 3 1 2 Toolbar Run Wizard Menu bar Run gt Wizard ck Ss 8 Open New Wizard Template E irn li ein fio ratio d Scouting ook in CJ Methods nd Templates mobilization Kinetics Linked Reactions a Kinetics Mass Transfer say 2 Ki netic sfida iid Surface Preparation Immobilization New Methods and Templates
170. 85 333 021 15 0 712891 185 14 0 411133 187 332 999 14 0 701172 187 13 0 400391 197 327 708 13 0 618164 197 12 0 310547 200 325 22 12 0 567383 200 11 0 216797 202 323 455 11 0 496094 202 10 0 198242 0 349609 9 0 182617 9 0 414063 8 0 25293 8 0 391602 i 0 181641 7 0 267578 6 0 143555 6 0 24707 b 0 0888672 5 0 15332 4 0 178711 4 0 170898 3 0 0712891 3 0 0576172 E 0 048828 0 0605469 0 0195313 1 0 0 0 0 0 0 0 0283203 1 0 0517578 2 0 0820313 2 0 102539 3 0 169922 3 0 194336 4 0 130859 4 0 303711 RPoint_Y 9 Beta2micro hi 3 0 0576172 0 18 0 74 0 102539 siz 332 645 16 0 60 332 791 15 0 53 332 898 14 0 501 327 761 13 0 421 329 21 12 0 35X 323 496 11 0 45 10 0 431 9 0 33 8 0 436523 6 0 204102 ES 0 126953 4 0 122 3 0 097 2 0 0 0 0488281 1 0 11 2 0 1 3 0 1 4 B 1E dz 185 B C 1 Exauationlkinetics Affinity Name Beta2 micro 2 3 4 Sample Beta2micro 5 Temperatu 25 8 Curve Fc 2 1 7 8 Model 1 31 Binding 9 Description 11 Curve ka Q MsS kd ds KD M 13 Ovcle 5 2 nM 14 Oycle 6 4 nM 15 Oycle 7 8 nM 16 Cycle 8 16 nM 17 Ovycle 9 32 nM 18 Ovcle 10 8 nM Rmax RU Conc M tc 12 1147601 0 002635 2 3E 09 24 50454 1 17E 09 2E 09 4E 09 8E 09 1 6E 08 3 2E 08 8E 08 Flow iulm kt RU Ms RI RUD co
171. A contact time 60 120 Inject B Cancel Eject rack tray Hy Menu bar Commands Eject Rack OK Inject command s OK Te Biacore T200 Control Software regeneration check blr ilz File Edit View Commands Run Tools Help bx Ed E i 7 2 Cyde 1 Curve Subtracted Fc 4 3 Lock scale hinding_1 EE baseline 1 1500 50 100 150 200 300 350 Time Window AbsResp SD LASD Slope RelResp Baseline ld Keywords in cycle 1 Value 5 1981 4 0 04 0 04 0 01 0 0 Yes baseline_1 5 4238 7 28 54 018 1526 2257 2 Mo binding 1 5 43265 381 021 2 03 23451 No stability 1 Flow 30 Flow Path 3 4 Online COMI Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 6 min
172. E RHIUN C BBESR CaRAE LIE NDUSETE C RA 5 T Regeneration Scouting Experimental Parameters Regeneration parameters Flow rate 30 pl min Stabilization period 30 s T Regeneration Scouting Results High viscosity solution Trend Chart Sensorgrams Experimental design amp Sample Response Baseline Number of conditions Number of cycles for each condition Settings Condition Regeneration solution Contact time s i Gly HCI pH3 0 30 2 Gl HCI pH2 0 30 3 lyHCI pH1 5 30 4 50mM NaOH BE Display Sensorgam lst sample cycle Conditions EEEE Sensorgram Fc 4 Application Wizard Assay Development Surface Performance 400 Te Surface Performance Injection Parameters Sample Solution mlgG ll Binding stability Contact time 60 s Flowrate 30 pl min Curve Name Fc 4 3 P 4 Assay Step Purpose Sample P Cycle number lt Hverlayy Binding stability E Zoom Lock X Value 4 Y Value Regeneration r 2 268 1 zolution d a
173. GE Healthcare 9 Biacore Life Sciences Biacore T200 version 2 Instrument Handbook GE imagination at work 1 1 1 ae 1 IM DB7 5 1 1 1 2 kk 2 1 1 3 4 1 2 kk 5 1 2 1 zd i kk 5 2e2 9 A a 10 1 2 4 GE ODEA A LD Fa ri 11 JE 1 U 14 2 1 a A IP ODE a D a Dy KK 15 SIME s SUB ERN RR RERO 18 2 12 199 ve ove 2X ES CDS DU recen iat ree E Dee eee erat t E e eet 20 EN VODI e E 22 2 2 2 e UO E E ETE E A A ii 22 PCM ia 0p m1 EEE EE A AE A A E AEE ii 22 AE E E a E A A 23 39250 RI 25 3 1 1 0 MO 0R KK 27 35152 7 Z s 3 Jb COD BIET este it ette reti Certe emer te hee Lee rest cti 34 ESCAPE iran E E IC ved TELS 41 4 QZZI PIWIE d S18 amp TEFHODSRTERSJ 45 Biacore T200 5 AIFA AE aire aN AN RA a a i 53 5 1 54 G
174. Kinetic constants are within instrument Kinetic constants are within instrument Kinetic constants are within instrument specifications specifications specifications Kinetic constants appear to be uniquely Kinetic constants appear to be uniquely Kinetic constants appear to be uniquely determined determined determined o No significant bulk contributions RI found o No significant bulk contributions RI found o No significant bulk contributions RI found Check that sensorgrams have sufficient Check that sensorgrams have sufficient Check that sensorgrams have sufficient curvature curvature curvature Examine the residual plot Pay attention to Examine the residual plot Pay attention to Examine the residual plot Pay attention to systematic and non random deviations systematic and non random deviations systematic and non random deviations F Allsen 3 Window gt Tile Horizontally Tile Vertically Evaluation Explorer Biacore T200 5 83 5 15 Multiple Rma
175. O Yes baseline_1 N 580110 200 014 106 221150 358952 054 ODEO 004 08 o binding_1 No stability 1 Flow 30 Flow Path 1 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 6 min I se I6 U TSERE USE NIUU S S Biacore T200 20 2 2 1 2 GU B 2 3 Fej Time window AbsResp SD LHSD Slope RelResp Baseline Id AS 1 1320 5 359815 008 10 000 0 0 Yes baseline 1 1 1370 5 596026 2 58 023 138 22 7210 No binding 1 1 2120 5 358 37 0 16 014 0 05 1 9 No stability 1 Id baseline 1 10 binding _1 5
176. Ositive control Control sample 96 Deep Well Microplate i 148 Positive control Control sample i 148 Positive control Control sample i 148 Positive control Control sample E 9 Q Q Q Q Q Q Q i 148 Positive control Control sample 00000000 148 Andyte Sample i 148 Analyte A Sample 100000000 E AOBRRRDBRBBBB5ll5l1 g 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 148 Analyte A Sample 440 ne Menu 73 5 Automatic Positioning l T Automatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Orientation Anchor Rack Vial Sife Pooling First Sort By Control sample 7 Cyan Column Bottom left Sample Small Ascending Sample Bl DarkBlue Column Bottom left Sample Startup II Crimson Column Bottom left Sample Wash E vellow Column Bottom left Reagent Small Ascending Small Ascending Large Ascending Small Content Ascending Y Y Y Solvent correction buffer 4 Il Blue Column Bottom left Reagent gt Pooling
177. Resp Baseline Id Keywords in cycle 1 Value 37 0 3b4B7 5 27 26 3 21 14 43 HN No PreConc1 Chip CM5 43 0 35567 1 32 90 0 62 17 58 HN No PreConc2 Ligand antibody 57 0 35828 7 35 23 0 28 18 83 HN No PreConc3 Method Amine 87 0 353833 33 31 053 17 81 HNA No PreConc4 Procedure TargetL evel 132 0 371328 29 70 0 28 15 88 HN No PreConc5 TargetLevel 531 0 3656384 0 10 0 02 0 05 0 0 Yes Baseline 1 1114 0 367378 317 0 80 1 65 333 No EDC NHS 1185 0 36808 4 0 06 0 06 0 02 110 0 No Fulse1 1258 0 368821 0 21 0 02 011 1837 No Pulse2 1332 0 36377 0 0 23 0 04 012 2785 No Pulse3 1382 0 3683330 0 20 0 06 010 300 6 No Pulse4 Online COM1 Temperature 25 00 9C Sensor chip CMS m o 4 4 4 4 4 4 4 4 4 4 4 cn cn cn can can an aa cot coi oi o Sample compartment temperature current 25 C set 25 C Running standby remaining time 4 0 days Te immobilization Results IEhip CM5 Response Response Flow cell Procedure Method Ligand Bound RU Final RU Target Reached 4 Target level Amine antibody 240 1 353 2 Yes Immobilization Results RU Target Reached Response Biacore T200 44 3
178. S 57 elea ges 2A YTA I ouk um ERAN Cotcete otc ecce eadem ILLAD e tI MCI S LATE 66 eo SP EE S I RE 87 5 2 93 CROSS ERE URN PRETERITO 94 5 2 2 kk 109 6 kiki 124 6 1 kk 125 CE EN AYY O E a A 126 6 3 DIA A DE a 147 TF FSF 151 THIS ris C UP o a 154 Tel b De 154 FASES DeSO pana Sanie anana a a NE 155 Tele ScEImptv BOHTOE TUDING naaa A rotis ee OE AE et 158 1 155 V asm BuUNer TUDNO ee ci 160 ITI OM E Nii 162 Teea l NOMO NZE kd C 162 23 7 AT ET i 163 8 34 019 425 TEE 166 8 1 166 8 2 gt OD a O a S AEA jl 166 8 3 167 Biacore T200 9 169 9 D2 EM ol AA A A fi 169 2 DO OG cc GA i 170 9 3 t e de iE E busto fb v ei Pedir Pret es En 172 Op qp tec Se ecco EXHI LRL RI LR AIL AM ERI C LR REEL EI 172 0 3 2 STET DEZ IRSE endi uec 174 9 3 3
179. SA MIB eps UM Lus E E A 53 575 159 L OW amp CON pO aaa 99 136 M Kontra No 1415 Moihod VONDE eerren E i i i 139 METES c E E E E T EE ITEM 14 Metheds and Telmbldles uada cat uet ar sta meom acit smt sp meses bdo to 28 31 34 38 41 57 64 106 125 148 AE T ee 137 IIl EIE ROK CA OTET 83 N RIE AEA EEEE TN 5 1 23 25 26 55 55 56 58 40 44 WEIT RB ro P2 OPE cid 29 37 61 103 147 162 179 NUO elk e cic EE LE 59 EiUIaners dre acc DC SNA 96 97 131 134 142 O Geni MO EA AA A AA AA AA A A A A A A A T 86 S A MM M M 94 126 P Predena NERONE RS 137 SIRAC EE 9 29 37 61 103 147 155 154 157 163 PhD 22 UDO pa RS OO 150 151 Biacore T200 Q ual asSSSSSIWE Euseb uonte EL E EL IUE E EL T OLLI IDE ELLE OUE ESL LIUIUS LL LIE 92 Te TE COMTO lesar EE E IR 73 116 R ROCK IEO i i EEE 12 13 30 38 64 106 148 NNOIGREINUID bee ee ei 12 podoenb Te i i i i i i lil 12 Aim Idus CC i Ni 151 132 154 142 fiscal AI AE TE E ii i i 20 DOISIeBeedBlBeeccou hec NS 20 51 RCN BIMIS p EOSMTRMAMETOH OM E REPRE 50 58 98 99 136 138 Redeneratom Cod daro sanae a bi 52 RENOVE SO EUO PR NS 70 113 pepe atasca e A iii i ll 152 heDOLl elec su teu Li MU Ni LM LL uel UL EE 1 5 119 REDO EDOM METER e 2l 175 176 BS RR M E M i ih hii 55 90 RU i SIUE SELLOS DELL QU DELE DELE 74 76 117 119 RSSIDONSGIBOUI oot cd MM M LO Md DM E 40 SS1 616 6 1S1 LO 40 RO UEI EC CN c i i ETOR 183 R
180. SE 155 163 DE Okaa EROR 58 95 128 137 DS cm TER 59 99 99 196 DM tu LU EIU UU M M MU LM E EM QU 5 26 46 128 Ble E S CD SA GR 67153 E ERC aE P OM 25 56 58 40 ROGER 11 30 38 49 64 106 148 Silsreud a vofel AM Ve a i ET NR DC REC RIEN TR 11 30 38 64 106 148 EMD Rr TADIA RE mtm 158 164 BO TE E 22 51 EDU nEROELON SIL i nM DELLI SUI S IE ELO DUE D ELAQUD DEL 22 51 EROBESIDBegec uu Da MM A E 157 Evauaton VON c NN 199 EXDOM CUVE S 182 Pra wash olere tHon Wi NE 98 137 F FOW BO 15 28 47 58 98 99 101 136 144 EO I PNE ERN RN i 15 29 47 59 98 99 156 HEC a co A LU LA Mu i UU UE 157 G Cae i ETE 138 aN EM a EE TT E NET A T AO 94 126 H FHeterggeneous ANAT RC E E L2 155 wi dizife 10 1 0 0 Tero RIS MN i EZ BI Pion DSO ON CA 136 allo esc Eo MEMO i i i 59 158 JEN TERRI IE ERN NT NP 138 Biacore T200 immobilizati oh PH SCONO oid cuti God i M ROMS Cu DN URINE Gta uis 26 27 28 52 53 HYIESOBIMZeUSResllbsroecsu e OtccGuE i i i A 43 DEPO NC AE TATE AE AA DM AEE EN EE AE T EAE D 53 E ECON I 18 19 48 49 ecta ndRecCOV ei 138 K Kem rut IM DM A MAL A 5599 15 15 7 05 95 116 118 120 E SE i 54 55 73 75 77 85 93 116 118 120 ee 45 54 55 56 75 91 92 93 118 A E RE S EETA EAA DIE AM UM EAE A AT 66 87 109 ISI EAN E AE E T E M MM E AEE E 85 Ee He
181. T EN Cycle 1 m Curve Sensorgram Fc 4 ProtreinA 20ug ml pH5 NHS EDC 2 Ligand 3 Ethanolamine 1 EDC NHS 200 400 600 1400 1600 Lock scale E immobiizea Fc Time window amp bsResp LASD Slope RelResp Baseline 4 277 0 5 359025 0 07 0 02 0 0 Baseline 4 830 0 5 350287 D 0 04 0 05 126 1 EDC NHS 4 1323 0 5 374125 O03 047 1503 3 Ligand 4 1813 0 5 374155 0 04 021 1513 8 Immobilized Keywords in cycle 1 Chip ContactTime FlowR ate Ligand Method Procedure Value CM5 420 10 Protrein 20ug ml pH5 Amine Time amp ndFlow Online COM1 Temperature 25 01 9C Sensor chip CMS Sample compartment temperature current 26 C set 25 C Running standby remaining time 4 0 days RU De immobilization Results Chip CM5 Response Flow cell Procedure Method _ Ligand Bound RU 4 Time and Flow Amine Protrein z ug ml pH5 1383 8 Response Final RU 1513 9 Biacore T200 40 3 HE 3 4 Response Bound Response Final 2 Bound Final NHS EDC
182. a CO A E E DR LU Dee 557 jm CP TEES 15 175 76 77 79 116 118 119 120 122 ndisse top Cue tse a Dc E cr ee 24 55 73 75 77 79 91 116 118 120 122 Pe a i 4 15 S AMPE Ond reden TOCK irn e S S 12 odiplexcormpartmenttenmpebabe astute cete it on ER tct cn Rn c i ata 29 37 61 95 103 128 SE E E IET IT EAIA A A AT E ATE T E E M 136 E uu E ME MU uu 76 119 9eNSOONIDNMGINNGNGNIGBINNANA acil aa ectetur acted boot d i 15b 152 155 SensorgralAd US UTIBDIE i 175 177 178 SOWANG UVE SC Ch NN i 48 MOVE OGAR SR GC GS 68 88 111 Snow CU ES OR SMMET VO NN CC 48 SOW OMEN CV 48 Biacore T200 SINOIECVEIE RI E DU EUN 98 156 III 68 88 S EEEE E E E MEER REM Et 94 AAC OTO O 138 Speci contact time dnd ow FATE REE NR 35 GAZAO PEN O m TE TOR TOR OT EE EE 59 98 157 eo EID EEA E E OE DERIT 76 119 SONED O W stn it es OR d uenia 6 9 22 22 39 51 05 107 148 155 154 157 162 166 OT RENE ii i 58 96 99 102 eda A GN CN NNS 90 SOCR e 65 108 150 EEPE Orm NO a Tm 52 SUIC adis See ANE T E E TU 28 354 41 ETE ud DM Du D LM DM MM D M D MC D M M M 1635 T OR6 16 SV 42 BDO NN TNR EE i i i ik 4 10 29 57 61 105 eNO E E EE DD E E 53 59 TS EHE DEZ OR N deer o Maca cc oU AU UL MO M MA ULM MO EL MM 82 GM EEC 82 TWS Re E 72 115 VO LL E I Lu MILL uM MUI E 99 Q5 c
183. ample 2 Sample 88 sample 2 Sample 88 sample 2 Sample DOOOOOQO0 e OO0OOOOOOOO0 ODOOOOOOO00 mercem mnc U 6 5 Menu gt Export Positions D Menu gt Simple Position Import Biacore T200 148 6 Eject Rack Rack tray port l OK
184. antigeni antibody 1Bug ml pH5 antigenz antibody 10ug ml nH5 antigen3 teratian 7 Chr 0 307 RUF Mas relative change 4 50 RIit 2 0 8337 Accept Curent Abort Current Abort Remaining Biacore T200 82 5 Biacore T200 Evaluation Software Kinetics antigen antibody blr i View Evaluation Tools Window Help e Sensorgram Plot asi Bar Chart A3 Kinetics Affinity Concentration Analysis Thermodynamics melna E cd antigen1 TER AEE BAA AEEY x Remove v Edit Curve Fc 2 1 Ligand antibody 10ug ml pH5 E Curve Fc 2 1 Ligand antibody 10ug ml pH5 Curve Fc 2 1 Ligand antibody 10ug ml pH5 F Sensorgram j All sensorgrams Fits 1 1 1 Binding v Fits 1 1 1 Binding v Fits 1 1 1 Binding v E Plot Baseline Sample Binding level Binding stability Binding to reference Report Point T able mm Report Point T able Kinetics Affinity J antigenl l A antigen2 AJ antigen3 10 5 100 50 0 50 100 150 200 250 300 350 100 50 0 50 100 150 200 250 300 350 100 50 0 50 100 150 200 250 300 350 Time s Time s Time s TT Quality Control Report Residuals Parameters Quality Control Report Residuals Parameters Quality Control Report Residuals Parameters
185. atus bar 8 2 Toolbar Eject p Menu bar Tools gt Eject Chip Biacore T200 A This will eject the sensor chip Eject Chip Biqcore T200 control software Biacore T200 Biacore T200 8 167 8 3 2 Dock
186. binding binding bility kotty abilty stability fi binding 3 binding x xtabilitysrsta Fbaseline e x binding stability 50 50 100 150 200 250 300 Time 0 baseline Sensorgram window _ eds Tools gt Sensorgram Adjustment Blank Subtraction A Enable Blank 5ubtraction Biacore T200 178 9 Blank Subtraction Enable Blank Subtraction RU Adjusted sensorgram amp binding ability hinding ability g5 ty x nding nding bi amp stability amp sabilitystability sites Response 0 baseline tpinding bi 2 E Pv 100 50 50 100 150 200 250 300 350 Time 0 baseline 9 3 6 100
187. ck Rack tray port l OK Eject Rack Tray Rack Positions Next gt l Te Method Builder Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according to the Rack Positions setup Fwials should be sealed with rubber caps and microplate with adhesive foil Place the buffer s an the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 1 h 38 min e cluding conditional statements temperature changes and standby Flow Estimated buffer consumption i A Buffer At least 100 ml plus 65 ml day for standby after run Start Save as Methods and Templa
188. d Builder Method Builder Method Builder Madified 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 3 28 2008 Show importable wizard templates Single cycle Kinetics Open Method Builder D Main Overview 6 General Settings Biacore T200 5 95 T Method Builder Main que General Settings 2 Data collection rate Detection Sample compartment temperature Hz C Vary with analysis temperature Assay Steps Cycle Types Variable Settings D Miscellaneous 5 e Buffer settings Concentration unit veicon M v Reton mem ETT After run Specify analysis temperature after run D Data Collection rate 10Hz 2 Detection 3 D Single DuqlMult Singe L 2 5 4 Dual L 354 d 8 5 Multi 12 544 2 1 4 3 2 1 5 1 4 1 Sample compartment temperature
189. dimm Response 0 baseline x stabilit ability Sestiitilitby Fabilty stability c Fej pn B5 gt R v E b 8 DE 0 d S 2 v 5 50 0 50 100 150 200 250 300 Time 0 baseline s 9 3 4 RU Adjusted sensorgram 450 350 cn oe Response 0 baseline 150 100 100 200 300 400 500 600 Time 0 baseline s Biacore T200 9 177 Cut RU Adjusted sensorgram c ability c amp c E amp stability eS ly ding inding bin sability stability Ae Response 0 baseline a CA c T amp m P 100 50 0 50 100 150 200 250 300 350 9 3 5 RU Adjusted sensorgram 30 Response 0 baseline a m imdimm
190. eline 10 Before binding 5 Before E stability 10 After ra baseline 10 Before E E ra binding 5 Before BE rn s ahility 10 After Before After Start of End of Inject Window Baseline Biacore T200 Start of Sample 1 Yes End of Sample 1 5 Ma End of Sample 1 5 Ma Start of Carrv oaver control 1 5 Yes End af Carrv oaver control 1 w 5 Mo End of Carry over control 1 5 Ma cen Cw cm Start of End of Inject Start of End of Inject Inject RU 5
191. ep purpose Assay Step Name Cycles Assay Step Startup 1 Startup Startup Sample Solvent correction N Startup Startup w Startup Startup Control sample mox Solvent correction Control sample Control sample Solvent correction Control sample Control sample Solvent correction LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics Solvent correction e Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Solvent correction Solvent correction LMW kinetics LMW kinetics v Cyces Assay step Startup 1 Sample 7 Solvent correction Cycle Run List Simulation Tool Control sample Control sample Control sample Control sample 1 Control Sample 2 Enter the number of cycles you plan to run for each assay step in the left panel The list of cycles for the run is displayed in the right panel Cycle type LMW kinetics Cycle Assay stepname Assay step purpose Startup Assay Step Name Cycles Assay Step 1 Startup Startup Sample 7 Solvent correction 1 Control sample wlm Startup Startup Startup Startup LMW kinetics LMW kinetics Solvent correctio
192. eport Point T able Reparan T able Kinetics Affinity am antigen Toolbar AJ Kinetics Affinity x RI 0 RU Residual Residuals Y B Z 1 lt 2 RU Chi
193. ers tms RU flet ce qu amp 2 n5 0 02560 Finish X NEKE M Ko M FE REXE A Rmax RU offset RU X 0 Y Chi RU Evaluation Explorer Toolbar AJ Kinetics Affinity Biacore T200 92 5 5 19 Quality Assessment Kp 1 2 232 FOIRETSG Rr Ko
194. hod name Copy of Amine Command Solution Contact Time sl Flow Rate ulimin w T PRE CONC Specified in Immobilization Setup MIXINJECT EDZ NHS 5D 50 I Inject P A WASH Ethanalamine Mig amp Inject LIGAMDINMJECT Specified in Immobilization Setup P IMJECT Ethanolamine 420 10 OK Biacore T200 3 37 LES Immobilization System Preparations Frime before run Normalize detector Temperature settings Analysis temperature C Sample compartment temperature C Prime Normalize Temperature settings Analysis temperature 25C Sample compartment temperature 25C Next gt inmmobilization Rack Positions EM EE EM EE Immob Fr 4 59 i MHS Immob Fc 4 I Immob Fc 4 Empty EDC MHS min capacity 124l 129 Ethanalamine Imrnnh Fc 4 a8 Pratrein 2 ug ml pH5 Immnh Fc 4 U Biacore T200 38 3
195. iable Set property as variable Sample 1 Solution Sen me gm L O Carry over control 1 4 Contact time eo s C Contact time s Dissociation time s Dissociation time 500 e n 80 s C Flow rate l min u Flow rate i C Estra wash solution Flow path Predip Extra wash after injection with 50 22 DMSO C Stabilization period o s Cycle types 4 6 3 Commands Report Paints Report Paints Capture Sample Enhancement Regeneration Commands Sample Larry over control L arry aver control 1 Solvent correction Inject amp ndHecaover General IF then Insert A inset Biacore T200 136 6
196. iii 10 29 57 61 105 129 APEE OUOMWIZO EET T DR 14 Asco Step DRODOFOOFIS atis tait cad eaten te tii te adeste odo ttn ioa tet A tese RDUM t RD DUI 151 142 Bosg EDS t uEU LU LM uL M I M M uH IE OU ULM 95 124 127 Po Face PO SIE DEI oda onte scd bum amba me pDR DUM E EMEND E MM 65 105 149 B BS EN 51 BaS INE cud uM i 2167 119 171 COO Oo ML en E EE E 68 81 88 Biacore Maintenance d m 152 154 155 160 162 163 EB AO SIS escasa Gi TU C ORIATUR 53 Slate Re c Mere CE CEU CLE TUE 67 110 171 BAN ele el OR oua guests bcd denas niae esu ues rcs cs tU c UL p cs UI cs reU 67 110 171 EIOS Ie eme E RI 72 115 BIGNRIAMNODNZO ON i bi i i 35 BRN m T 95 129 DU E uiu uL i 45 C COD UE E i i D EOE ii 58 136 137 NO 58 E D ATE E A ATES T A A AEE OAE AA A 75 76 91 118 119 SOR AO Ne 53 60 139 CONCER O NOE SR 53 E E E aet eM UM UM M M M LL 95 129 Concent atone pel OU CIS aestate aaa Pt I Lead I Mut S ead I Ue ce UI 98 Gera c m ore el DE T U T31 Gees oa 29 49 59 98 99 136 EE ACI DET ATE ASE E AAEN IM I E M AL ET 182 CIO cM MM M Uu I 45 UTERE NM RR T 78 80 121 123 Biacore T200 CO Ne SR i i 35 Gu RU E DE 61 155 134 VC HRN 2 RR RR 97 127 D DO OCONEE MN E E 95 128 SS eee cH 154 DSO ON
197. inetic with recommended 5 concentrations ie number of injections in each cycle Vehcaion 1 Sample 1 Regeneration 1 Heb Sample Type Concentrations per cycle contact time Dissociation time Flow rate Flow path Extra wash after injection with Stabilization period Settings for Sample 1 Type Single cycle kinetics Method Variables Evaluation Variables Set property as variable Sample solution Is variable Concentrations per cycle Bohn caen Contact time 120 s datis sed Dissociation time le00 s Flow rate 30 plz min Flow path Boh w M Extra wash after injection with ES SNabilization period Close Single cycle kinetics 5 s s ul min 30 ul min Both Multi
198. ion btL179U TwZ7d4d4027 7Z7 kELR Tools gt More Tools gt Maintenance Tools Normalize Start Hormalize This procedure normalizes the detectar signal Total run time i about 8 minutes Required olution fram Maintenance Kat Bl amp normalizing solutian Next gt l Stqrt l Harmalize Normalizing please wait Time left 00 08 16 Normalize The Normalize procedure is completed Close Standby flow Biacore T200 7 163 7 3 Desorb and Sanitize 1 Biacore Maintenance Kit type 2 BlAtest solution
199. ip HPA 3 BR 1005 33 Series S Sensor Chip L1 3 BR 1005 38 His tag Series S Sensor Chip NTA 3 BR 1005 32 Biacore T200 1 9 1 2 2 ZY ZYJE RE kD FHE Menu bar Tools Prime Tools Help Prime Shutdown Stqrt Prime Place buffer on the left hand tray and insert tube A Place water on the right hand tray and insert the water inlet tube Prime Prime Friming please wait Time left QD OB 46 The Prime procedure is completed Close Standby flow 1 8 Prime Biacore T200 10 1 1 2 3 Analysis temperature
200. l Sample 1 Thermo 1 16 30 Biacore T200 134 6 6 2 Recurrence Number of replicates Cycle Run List Er Genesi Satin se oceres Variable Settings ooo a me u Recurrence Number of replicates Cycle Run List Cycle Run List Simulation Tool Enter the number of cycles you plan to run for each assay step in the left panel The list of cycles for the run is displayed in the right panel Cycle type LMW kinetics LMW kinetics LMW kinetics Cycle Assay stepname Assay st
201. n Solvent correction Solvent correction LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics LMW kinetics Solvent correction LMW kinetics LMW kinetics Control sample Control sample Control sample Control sample e Sample Sample Sample Sample Sample w Sample Sample Sample Sample Sample Sample Sample CN Sample Sample Solvent correction Solvent correction en Control sample Control sample Control sample Control sample e Close Recurrence Number of replicates Biacore T200 6 135 Cycle types T Method Builder Main Cycle types Description of selected cycle type Solvent correction This cycle is used in startup sample and control sample steps LMW kinetics Contains injection of sample and carry over control running buffer Ccewes 12 Variable Settings Commands Report Points 9 Tupe High performance v S Method Variables E valuation Variables P Inset X Remove me x Sample solution Is var
202. nce Chip For this procedure The surface on other sensor chips may be damaged by the solutions used Last run time 5rz r2 008 9 25 AM Biacore T200 152 7 Biqcore Maintenance Kit type 2 BR 1006 51 BIAdesorb solution 1 95 ml x 2 BlAdesorb solution 2 95 ml x 2 BIAtest solution 65 ml BlAdisinfectant solution conc 10mlx3 BlAnormalizing solution 90 ml HBS N Buffer 10 X 50 ml Sensor Chip Maintenance 1 BlAdesorb solution 1 4 _ C SDS BIAdesorb solution 1 4 C Sensor Chip Maintenance Dock Biacore T200 7
203. nlet tube BIAdesorb Solution 2 25 ml 15 ml 2 A BC D BIAdesorb Solution 2 25 ml BIAdesorb Solution 2 15 ml Start 2 3 lesnrh and Sanitize Step 3 Wipe the pump inlet tubes with a moist tissue Flace 50 ml diluted Bl amp disinfectant Solution an the left hand tray and insert all four pump inlet tubes Place 30 ml diluted Bl amp disinfectant Solution an the right hand tray and insert the water inlet tube BlAdisinfectant Solution 50 ml 30 ml 2 A B C D BlAdisinfectant Solution 50 m BlAdisinfectant Solution 30 ml Start Biacore T200 7 157 3 4 Desorb and Sanitize Step 4 Wipe the pump inlet tubes with a moist tissue Place water on the left hand tray and insert all four pump inlet
204. nsorgram Fc 3 amp 26 2 3iil Lock scale 5 10 15 Fc Time Window AbsResp SD LASD Slope HelHesp Baseline Id I Keywords in cycle 1 Value Flow 30 Flow Path 3 4 Online COM1 Temperature 25 00 9C Running manual run Sample compartment temperature current 25 C set 25 C Run time 1 min 4 2 View Show Only Current Curve 1 View Show All Curves View Show Curves of Same Type Inject command qe Menu bar Commands Inject Biacore T200 4 49 Inject Vial well position R2B1 s Minimum required volume in viral vell For this injection B8 pl Vial well position 5KO A
205. oor fit RU Residuals for a good fit RU Biacore T200 118 5 Report ES Kinetics Affinity Fit Kinetics Create Curve Fc 4d 3 Ligand antibady Sample antigen Temperature 25 C Add Fit Current Fits Model 1 1 Binding 1 1 Bindini ida Fi Lu T B 5 4 3 2 1 1 2 Quality Control Report Residuals Parameters l 225E 6 0 003754 3 065E 39 4 415Ec11 1 063E 9 30 00 1 372E 12 0 2948 e 125E 9 0 2917 4 250E 3 0 4467 B 500E 9 0 2712 1 700E 0 4539 ka 1 Ms DOREEN ka 1 5 FERE TER ES XE EL Ko M FERE AE AN Rmax RU RI RU bulk effect Chi RU U value U XU 3 14 Binding Finish Evaluation Explorer Biacore T200 5 119 7 Sensorgram ri All sensorgrams Flot a Baseline Sample a Binding level Binding stability g Binding to reference 7 R
206. orrection Assay steps New Biacore T200 142 6 Mew ras elete Copy T Move Up T Move Down Cycle Run List Azsay step properties Base settings M ame Control sample 4 t Control sample Control sample 1 time as entered Every 15 cycles Startup 5 Startup Thermodynamics 3 times as entered Thermn 5 5ample Thermodynamics 1 time as entered Control sample 5 t Control sample Control sample 1 time as entered Every 15 cycles Assay step 1 Mot connected 1 time as entered Recurrence Repeat assay step within Connect to Mot Connected v cycle type NN Assay step 1 Assay step 1 Base settings Base settings cycle type Name Solvent correction Purpose Solvent correction Connect to cycle type Solvent correction Recurrence Number of replicates Assay step preparations
207. r E gt Report Point Table m Report Point Table Evaluation Explorer gt Work area Menubar gd NCOTFEQvZ7F amp amp dco ife x 31 3n Toolbar Evaluation Explorer ooram pot Baseline AbsResp Binding level Baseline RelResp Binding stability Baseline RelResp Binding to reference Binding level Work area Evaluation Explorer Biacore T200 172 9 9 3 Evaluation Explorer Sensorgrum 4 JLI 73 5 All sensorgrqums Work area Sensorgram window 9 3 1 Sensorgram window Cure Name Fezz 1 sj lt Assay Step Purpose Herlan Cycle Overlay h 4 Curve Name Fe 2 1 J Jo i eal
208. re after it is started Next gt Desorb and 5anitize Required solutians from Maintenance Fat Bl amp desarb solution 1 ane volume af 25 ml and ane volume of 15 ml Bl amp desarb solution 2 one volume af 25 ml and one volume of 15 ml Bl amp disinfectant saluton one volume of 50 ml and ane walume af 30 ml Next gt Biacore T200 156 7 hesnrh and 5anitize Step 1 Place 25 ml Bl desarb Solution 1 an the left hand tray and insert all four pump inlet tubes Flace 15 ml Bl desarb Solution 1 an the night hand tray and insert Ehe water inlet tube Back Start Close BIAdesorb Solution 1 25 ml 15 ml 2 A B C DI amp ZANT BIAdesorb Solution 1 25m EK BIAdesorb Solution 1 15m start 1 2 Desorb and Sanitize Step Wipe the pump inlet tubes with a moist tissue Place 25 ml Bl desarb Solution 2 an the left hand tray and insert all four pump inlet tubes Place 15 ml Bl desarb Solution 2 an the right hand tray and insert the water i
209. rime Normalize Temperature settings Analysis temperature 25C Sample compartment temperature 25C Cycle Run List Te Kinetics Affinity Cycle run list 2 Startup buffer E Startup buffer 4 Startup buffer 5 Sample antigeni D 11500 B Sample antigeni D 11500 Sample antigeni 1 1 11500 8 Sample antigeni 2 2 11500 9 Sample antigeni 4 3 11500 10 Sample antigeni 8 5 11500 mm Sample antigeni 17 11500 12 Sample antigeni 1 1 11500 13 Sample antigenz 11500 Next gt Biacore T200 62 5 T Kinetics Affinity Rack Positions Conc nM MW Da E 118 antigeni Sample 11500 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigeni Sample 118 antigen2 Sample 118 antigen2 Sample 118 antigen Sample 118 antigen2 Sample 118 antigen2 Sample 118 antiqen2 Sample 118 antigen2 Sample 118 antigen2 Sample 118 antigen Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 118 antigen3 Sample 334 buffer Startup 541 buffer Regeneration 1763 Gly HCl pH2 5 Regeneration
210. s Thumbnails Export All Graphs And Table Copy Copy All Graphs Copy All Thumbnails Paint WordPad Biacore T200 86 5 Table Include p Table Thumbnails On Off Rate Map Steady State KD Plot Tool mT Table Columns Sample ka 1 Ms kd 1 s KD M Rmax RU Control Rmax RU 100 Da Chi RU Ligand v antigen3 5 60e 05 2 04e 03 3 65e 09 108 7 NjA 0 112 antibody 10ug ml nH5 v antigen3 NjA NjA 3 59e 08 198 3 N A 0 0462 antibody 10ug ml nH5 v antigen3 NjA NjA 3 59e 08 198 3 NjA 0 0462 antibody 10ug ml nH5 v antigen1 1 53e 06 2 61e 3 1 70e 09 105 3 NjA 0 1 antibody 10ug ml nH5 v antinen1 NjA NJA 1 37e 08 165 1 N 0 517 antibody 10ug ml nH5 v antigen2 2 19e 05 1 63e 03 7 44e 09 114 3 NjA 0 0501 antibody 10ug ml nH5 E Biacore T200 Kinetics Summary 1 bme Fie View Help T1 m 37 Table Thumbnails On Off Rate Map Steady State KD Plot m Table Columns Selected E valuation Ligand Sample antigeni 90
211. se 50 2w927kLb4 amp BsciAUL OK amp 2Uv2UzS Eject Rack Tray Rack Positions Next gt Biacore T200 3 31 Te Immobilization pH Scouting Prepare Run Protocol Tahoma 1n Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according tn the Rack Positions setup ials should be sealed with rubber caps and microplate with adhesive foil Place the bufferis an the left hand tray and insert the correct tubingis see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle on the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 18 min excluding conditional statements temperature changes and standby Flow Estimated buffer consumption ad Running buffer J j j E At least 100 ml plus 65 ml day for standby after run EUCRSVGIERASIB AEA 53E RL LL7 RPBROeSDZ medueS Start Save as Methods and Templates 7 Bia Users
212. tes 7 Bia Users Don t Save Biacore T200 5 107 Save Results From Run s Save in e CSK My Recent Documents 3 Desktop immobilization of antibody blr E My Documents 43 My Computer File name SCK antibdy antigerl My Network Savesstpe Pesut fle b B Save in File name Sqave BiacoreT200Control Software SCK_antibdy antigen blr Ef IEG Fie Edit view Run Tools Help zum x RU Lock scale 545 544 200 400 600 1200 Time Window AbsResp SD LASD Slope RelResp Baseline Id Keywords incycle5 Value 51 8 176 8 131 8 364 7 379 7 552 8 567 8 740 7 755 7 5231 0 15 015 003 DO Yes baseline AssayStep Sample 5324 0 11 D 11 0 02 3 2 No binding AssayStepPurpose Sample 531 4 0 22 022 0 02 2 3 No stability Buffer Buffer 535 0 0 14 0 13 002 53 No binding 2 CycleType Sample 5341 0 17 017 0 01 49 No stability 2 Sample 1 Conc s 1 053 5378 015 015 4003 7 No binding 3 Sample 1 Conc H2 2 125 537 0 0 15 015 0 01 7 8 No stability 3 Sample 1 Conc H3 425 5405 0 15 015 0 01 11 4 No binding_4 Sample 1 Conc H4 8 5 5334 0 15 015
213. tion k 1 Running buffer j t least 100 ml plus 65 ml day for standby after run Start Save as Methods and Templates 2 Bia Users Don t Save l Save in File name Sqve Biacore T200 3 39 Standby flow 3 3 Ctrl Breakl T Biacore T200 Control Software Immobilization of PrteinA blr iz File Edit view Run Tools Help E S EE B
214. tion Anchor Rack Vial Sife Pooling First Sort By Control sample 7 Cyan Column Bottom left Sample Small Ascending Sample Bl DarkBlue Column Bottom left Sample Small Ascending Startup II Crimson Column Bottom left Sample Small Ascending Wash E vellow Column Bottom left Reagent Large Ascending 1 Small Content Ascending Y Y Y Solvent correction buffer amp Il Blue Column Bottom left Reagent gt ol Auto Poolingo_ Yes OK Automatic Positioning Biacore T200 64 5 l Eject Rack Rack tray port OK Eject Rack Tray Rack Positions Ne
215. ulmi Flow path Reference subtraction Detection in Flow cl 3 4 O a Flow path 1 E Flow path 1 2 Q I Flow path 2 e E Flow path 3 4 Q a Flow path 3 C Flow path 1 2 3 4 ES Flow path 4 Flow rate 30 ul min Flow path Reference subtraction 2 1 4 3 2 1 3 1 4 1 Rack Start ve Results From Run hs Savein 3 T100manual O B eg Immobilization of Prteina blr manual blr My Recent pHscauting blr Documents Desktop E Mu Documents hy Computer a File name regeneration check wv hiy Network Saveastype Result file bl v File name Save Biacore T200 48 4 Te Biacore T200 Control Software regeneration check blr ilz File Edit View Commands Run Tools Help il t En B n Cycle 1 o Curve Se
216. v Westem Sensorgram window Export Curves txt Excel Biacore T200 text Beta2micro highZ_X Beta2micro high2 Y RPoint X Excel File Export Result To Excel xls Evaluation Explorer Excel xls 19 0 558594 3 0 0712891 19 0 833984 18 0 533203 0 0 18 0 761719 0 17 0 5625 2 0 0820313 17 0 779297 2 16 0 457031 182 332 89 16 0 740234 182 15 0 417969 1
217. x 1 F Kinetics Affinity Select Curves Create Select evaluation mode Single mode Batch mode Curves Curve v Ligand Sample 89 w Temperature 37 Flow Contact Time Diss Time ul min s s 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 1 600 0 600 0 C Zoom lock Show concentration series Show blanks O O T F Kinetics 7 Affinity Select Curves Create Select evaluation mode Single mode Batch mode Curves Curve Fc 24 1 v Ligand Sample Temperature Flow Contact Time Diss Time ul min s s 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 1 600 0 600 0 600 0 600 1 600 0 600 1 Zoom lock Show concentration series Show blanks C Show average blank s Curve Add Rnmax 5
218. xt gt Te Kinetics Affinity Prepare Run Protocol PEE Tahama M 1 Prepare Run Protocol Make sure the correct sensor chip is docked Make sure all samples amp reagents are loaded in the rack and microplate according ta the Rack Positions setup Fials should be sealed with rubber caps and microplate with adhesive foil Place the buffer s an the left hand tray and insert the correct tubing s see below Mote Standby after run will use buffer A Make sure there is fresh water in the water bottle nn the right hand tray If necessary empty the waste bottle before start of the run Estimated run time 4h 28 min excluding conditional statements temperature changes and standby Fow Estimated buffer consumption j 3 3 3 Running buffer J j t least 200 ml plus 65 ml day for amp tandbw after run mc Start Save as C Methods and Templates 7 Bia Users Don t Save Biacore T200

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