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OPV-IL6WDR - Siloam Biosciences

1. PROCEDURE FOR MIGRATING A VALIDATED ASSAY FROM CONVENTIONAL 96 WELL MICROPLATE TO OPTIMISER MICROPLATE Page 18 of 35 SANDWICH ELISA ASSAY TRANSFER GUIDE The Optimier microplate ELISA procedure is a chemifluorescent immunoassay procedure in which traditional ELISA reactions take place within the unique Optimiser microplate architecture Briefly capture antibody is immobilized on the internal surfaces of the plate s microchannels Following a wash step any unreacted sites on the microchannel surface are blocked with a blocking solution Standards and samples are dispensed to the Optimiser wells Antigen present in samples and standards will be specifically captured on the microchannel surface by the immobilized capture antibody Following another wash a biotin labeled detection antibody is added to the wells The biotin labeled antibody will bind antigen that has been captured and immobilized on the microchannel surface thus sandwiching the antigen between the capture and detection antibodies Following another wash horseradish peroxidase labeled streptavidin SAv HRP is added to the Optimiser wells The streptavidin of SAv HRP binds specifically to the biotin moiety of the biotin labeled antibody if it is present in the capture antibody antigen detection antibody complexes formed and immobilized on the microchannel surface Following two additional washes a chemifluorescent substrate is added to the wells If horseradish pe
2. anti mouse IgG anti goat IgG etc Reagent must be titrated extensively to determine optimal working conditions e For using HRP directly conjugated detection antibody Reagent must be titrated extensively to determine optimal working conditions Page 21 of 35 Frequently Asked Questions Optimiser based ELISA Can I use Optimiser microplates for all the assay types that run on a normal 96 well plate The current version of Optimiser microplate has been validated for ELISA applications such as direct indirect sandwich and competitive immunoassays For other applications please discuss your application with Siloam s tech support What detection modes can be used with the Optimiser microplate e The current version of Optimiser microplate is well suited for fluorescence chemifluorescence mode detection e We will have a version suitable for chemiluminescence mode available in the near future e Absorbance mode does NOT work with the Optimiser microplate am not sure if the 10 minute incubation in room temperature will work I would prefer to incubate for at least 30 minutes or in 37 C Is that OK Actually NO We strongly recommend that incubation times should not exceed 20 minutes Incubating beyond 30 minutes or at 37 C will cause evaporative losses How critical is the 10 minute incubation window e Most binding reactions on Optimiser microplates saturate in 5 minutes Users can actually use even
3. Wait 1 second Withdraw the tip from the liquid 4 To dispense liquid hold the pipette nearly vertical With the pipette tips touching the surface of the Optimiser well depress the operating button steadily until the liquid is dispensed 5 Note The pipette tip must make contact with the well surface for proper dispensing see RIGHT frame below Do not pipet directly into the hole at the bottom of the well see WRONG frame RIGHT Ty D a Ka xa Figure 4 Pipette tip positioning for dispensing in the Optimiser microplate Additional Technical Considerations 1 The Optimiser system has been qualified with aqueous liquids only Do not use solvent containing samples 2 The buffer reagents provided with the assay kit have been developed and validated for the Optimiser microplate Do not substitute alternate buffers or reagents 3 The presence of particulates in liquids dispensed to Optimiser wells may block liquid flow through the microchannels a Centrifuge serum samples and serum containing tissue culture supernates for 10 minutes at 13 000 rpm prior to testing 4 Small flow rate variations time to empty well do not affect assay results Using Electronic Multi channel Pipette An electronic multi channel pipette is ideally suited for use with Optimiser microplates since a it eliminates possibility of injecting bubbles and b can be used for convenient repetitive load
4. selecting the optimal capture detection antibody concentrations Experiment 3 determining dynamic range of migrated assay on Optimiser microplate Figure 7 Schematic assay optimization procedure for Optimiser microplate Page 19 of 35 Requirements for Conventional Assay to be Transferred to Optimiser Microplate For transfer to Optimiser microplate a robust conventional 96 well plate assay is expected with the following minimum performance metrics e Reasonable background zero reading for a colorimetric assay the OD450 630 absorbance reading of background shall be less than 0 15 e g after a standardized colorimetric substrate development time such as 15 minutes e Reasonable dose response with various concentrations of standard for an absorbance assay the OD450 630 absorbance reading of highest concentration in detectable range shall be higher than 1 5 e g after a standardized colorimetric substrate development time such as 15 minutes In addition to satisfying the minimum performance metrics the following information is required for the assay transfer process e Known concentrations or dilution ratio for capture antibody and detection antibody working solution The capture and detection antibody solutions must be in a format such that at least 4x concentration solutions as compared to working concentration for conventional 96 well plate can be prepared e HRP conjugate the detection antibody must NOT be directly
5. system which can be detected using the appropriate excitation and emission settings Figure 5 Quantitation does not require filters that precisely match the excitation emission maxima However a non overlapping filter set with a band pass that includes the excitation emission spectra is required Wavelengths can be set at 530 575 nm for excitation and at 585 630 nm for emission Below are examples for different types of readers e Filter based readers Install 528 20 nm or similar band pass filter for excitation and 590 35 nm or similar band pass filter for emission e Monochromator based readers Set excitation wavelength at 544 nm and emission wavelength at 590 nm e Readers with pre configured optical set Select the wavelength setting for Rhodamine or Cy3 Fluorescence Solutions Preparation Serial Dilution of Activated OptiGlow Substrate In well A1 of a supplied 96 well v bottom plate add 50 uL of OptiGlow A 50 uL of OptiGlow B 5 uL of OptiGlow C and 1 uL of supplied SAv HRP stock solution mix well and wait for 2 minutes The substrate will be fully developed to a red fluorescence dye solution and stable for hours Prepare 1 2 serial diluted solutions with OptiWash to prepare 15 fluorescence solutions with 1 zero point 1 2 blank a Load 50 uL of OptiWash to well B1 H1 and A2 H2 Do not use other buffers b Transfer 50 uL solution from well A1 to well B1 and mix well c Change the tip repe
6. 20 minutes at RT 7 Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step 8 Dispense 5 uL detection antibody working solution to each well Incubate 10 minutes at RT 9 Dispense 5 ul OptiWash to each well Wait 10 minutes to proceed to the next step 10 Dispense 5 uL SAv HRP to each well Incubate 10 minutes at RT 11 Dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step 12 Again dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step 13 Dispense 5 uL OptiGlow working solution to each well Incubate for 15 minutes at RT a Caution Observe the wells during the incubation When the substrate has completely drained from all wells remove the plate and pad from the holder Discard the pad Wipe the bottom of the plate with a Kimwipe to remove any liquid on the bottom surface of the plate Step 13a will be completed within the 15 minute substrate incubation time 14 Place the plate in the reading chamber of a fluorescence plate reader Promptly at the conclusion of the 15 minute incubation read the plate Page 24 of 35 It is common to see slight differences in the time required for different wells to empty This difference has no impact on assay performance To facilitate work flow incubations designated as 10 minutes may be extended to 20 minutes with no impact on method performance Optimiser wash is perform
7. 30 uL Vortex mixer Microplate fluorescence reader and control software 6 Analytical software eS Assay Layout The plate layout of IL 6 standard concentration is shown in below Each concentration will be run in triplicate three columns of one Optimiser microplate will be used Table 2 Plate layout of IL 6 concentration pg mL for demonstration assay 1 2 3 A 3000 3000 3000 B 1000 1000 1000 C 333 3 333 3 333 3 D 111 1 111 1 111 1 E 37 0 37 0 37 0 F 12 3 12 3 12 3 G 4 1 4 1 4 1 H 0 0 0 Page 13 of 35 A Very small volumes of assay reagents are required and provided for Optimiser based assays A quick spin mini centrifuge is CRUCIAL to recover all material in Items 9 12 Use a spin each time the assay reagents are to be used Reagent Preparation The incubation times for Optimiser microplate are only 10 20 minutes Preparing all the reagents samples standards in advance will allow for proper timing especially for first time users Always prepare extra volume of solution for easy transferring Siloam suggest to prepare 30 uL extra volume each well in 96 well v bottom plate This volume can be reduced with careful pipetting if sample is very limited or precious Bring all reagents to room temperature before use and prepare all necessary dilutions before beginning the test procedure 1 3 OptiBind OptiBind H is provided in a ready to use form N
8. 5 e g after a standardized colorimetric substrate development time such as 15 minutes Page 20 of 35 e Conventional colorimetric assay Known information o Concentration of capture antibody 2 ug mL o Concentration of detection antibody 2 ug mL o Conjugate for detection antibody biotin conjugated o Assay dynamic range 4 pg mL 250 pg mL HRP Concentrations in Assays on Optimiser Optimiser microplates are an exquisitely sensitive platform for high sensitivity ELISA with minimal sample reagent volume requirements It is CRITICALLY IMPORTANT to follow the guidelines for HRP conjugate to ensure that the sensitive response from Optimiser is not overwhelmed by erroneously high HRP concentrations The use of a biotinylated detection antibody is recommended with the well characterized and validated SAv HRP provided by Siloam Biosciences Cat OMR HRP to obtain the best response e For biotinylated detection antibody a vial of appropriate SAv HRP is included in the kit Please prepare the working solution at 1 150 dilution with OptiBlock buffer It is strongly recommended that the SAv HRP provided by Siloam Cat OMR HRP be used for all assays on Optimiser microplate The concentration and activity have been characterized and optimized for use with the Optimiser microplate system Use of alternate SAv HRP may lead to low signals or very high backgrounds e For using HRP conjugated secondary antibody e g
9. Optimiser Microplate Adoption Kit also contains necessary reagents of an IL 6 sandwich ELISA Assay to demonstrate the capabilities of Optimiser based assays under controlled conditions by a user The representation of the expected data produced from Optimiser Microplate Adoption Kit is not intended to be used as a commercial assay kit this control ELISA is provided for training purposes only Materials Required for Demonstration Assay and Supplied with Optimiser Microplate Adoption Kit 1 One Optimiser holder Optimiser microplate 3 One new Optimiser pad single use One 96 well v bottom plate OptiBind H buffer OptiBlock buffer OptiWash buffer OptiGlow substrate kit contains component A B and C IL 6 capture antibody 10 Lyophilized IL 6 standard 11 IL 6 detection antibody biotinylated 12 Streptavidin HRP NU AWN Materials Required for Assay But Not Supplied with Optimiser Microplate Adoption Kit 1 Eppendorf or similar tubes for centrifugation and dilutions 2 Kimwipes or other laboratory tissue paper 3 Reagent reservoirs V shape reservoir 4 Pipette tips for delivering in the ranges of 1 10 10 100 and 100 1000 uL Equipment Required 1 Pipettes capable of accurately and precisely delivering liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipette capable of accurately and precisely delivering 5 uL Multichannel pipette capable of delivery of
10. The extra volume may be reduced with careful pipetting if sample is very limited or precious Bring all reagents to room temperature before use and prepare all necessary dilutions before beginning the test procedure 1 OptiBind Twelve types of OptiBind are provided in ready to use form No further preparation is required Do not substitute other coating buffers for OptiBind 2 Capture Antibody Use same concentration as conventional assay prepare the capture antibody working solution by diluting the capture antibody stock in 12 types of OptiBind to make 100 ul final working solution Dispense the capture antibody working solutions in different OptiBind coat buffers into a single row in the 96 well v bottom plate one per well 2 pg mL capture antibody concentration is used in the example assay Page 20 Hence 100 uL each of 2 ug mL capture antibody working solution in each of OptiBind A OptiBind B OptiBind C OptiBind L would be prepared for this step 3 OptiBlock For blocking step prepare 1 mL of OptiBlock in a v shape reservoir 4 High Concentration Protein Standard Prepare 1 mL protein standard with concentration of 80 of top standard diluted in OptiBlock in a v shape reservoir 250 pg mL of IL 6 is the top standard in the example assay Hence 200 pg mL IL 6 standard would be prepared for this step 5 Low Concentration Protein Standard Prepare 1 mL protein standard with concentrat
11. Withdraw the tip from the liquid touching it against the edge of the reservoir to remove excess liquid d Dispense the liquid into the loading well of Optimiser microplate by gently and steadily pressing the pipette s operating button to the first stop Briefly hold the operating button in this position e With the button in this position move the tip from the loading well to the reagent reservoir immerse the tip in the liquid and aspirate Pipetting step Ready position 1 2 st sto HHEH Second Stop Figure 3 Reverse Pipetting procedure THE USE OF PROPER PIPETTING TECHNIQUE IS CRITICAL TO AVOID AIR BUBBLES Page 5 of 35 A The pad must be oriented correctly with the smooth surface tape side facing the holder and absorbent surface touching the microplate A THE USE OF PROPER PIPETTING TECHNIQUE IS CRITICAL TO AVOID AIR BUBBLES Air bubbles will occlude the microfluidic channel and stop the flow of the Optimiser Accurate and Precise Delivery of 5 uL Volumes Assays on Optimiser microplate require the accurate and precise delivery of 5 uLvolumes The following guidance is offered to users 1 Use pipette for which the upper limit of their operating range is lt 10 uL 2 Use pipette tips appropriate for 5 uL pipetting 3 To aspirate liquid hold the pipette near vertical and immerse the pipette tip in the liquid to a depth of approximately 2 mm in the liquid Withdraw the operating button steadily
12. chamber of a fluorescence plate reader Promptly at the conclusion of the 15 minute incubation read the plate Page 28 of 35 To facilitate work flow incubations designated as 10 minutes may be extended to 20 minutes with no impact on method performance Optimiser wash is performed by simply dispensing OptiWash to the wells Caution In rare cases a well may not empty in 10 min If so blot the reagent from the well with a tissue Do not analyze signal from this well Selecting the Optimal Antibody Concentrations Use the signal noise S N ratio to determine the optimal combination of capture and detection antibody concentrations Example Data As an example an antibody optimization test has been performed for IL 6 assay Protein standard concentration is 50 pg mL Three concentrations of capture antibody 2 4 8 ug mL and three concentrations of detection antibody 2 4 8 ug mL have been used for this assay Use Biotek FLx800 fluorescence plate reader with excitation filter at 528 20nm and emission filter at 590 35 sensitivity at 45 Data acquisition and analysis utilized Gen5 software and Excel Results are shown as below Mean value of results Capture antibody concentration Detection antibody 8 ug mL 4 ug mL 2 ug mL concentration 1 2 3 4 5 6 7 8 9 10 11 12 A 8 ug mL 2707 337 2339 348 1650 359 B C 4 ug mL 2476 297 2097 233 1730 266 D E 2 pg mL 2344 238 2073 289 1543 292 F Co
13. detection antibody stock in OptiBlock in a clean plastic tube add 20 uL of detection antibody stock solution to 480 uL of OptiBlock b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate 6 SAv HRP The procedure requires 5 uL of the SAv HRP working solution for each assay well to be used a Prepare a 1 150 dilution of the detection antibody stock in OptiBlock in a clean plastic tube add 4 uL of SAv HRP stock solution to 0 6 mL of OptiBlock b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate 7 Substrate solution The procedure requires 5 uL of the working substrate solution for each assay well to be used a Prepare the working substrate solution no more than 30 minutes before the anticipated time for reading the completed assay b To create the substrate working solution combine OptiGlow A OptiGlow B and OptiGlow C in a ratio of 50 50 5 parts respectively in a clean plastic tube and vortex gently to mix add 250 ul of OptiGlow A 250 ul of OptiGlow B and 25 ul of OptiGlow C OptiGlow C is a solid in 4 C It can be thoroughly thawed at RT or 37 C to enable you to pipette correctly and for it to function effectively Warm the reagent in a 37 C incubator oven heater or by holding the vial gently in your hands c Dispense 60 uL of the working solution into each wel
14. no longer a limiting factor b The surface area of the microchannel is 1 5 times the surface area at the base of a conventional 96 well ELISA plate The volume contained in the microchannel is 5 ul leading to 50x higher surface area to volume ratio which allows for extremely efficient binding reactions 2 Even for the well that drains in 8 minute the initial section of the microchannel towards the center is filled up in 2 3 minutes Optimiser characterization data shows that the first few loops of the microchannel contribute 95 of the optical signal hence even if the last 1 2 loops take significantly longer to fill their contribution to the signal is almost negligible Consequently variations in signal from the last loops have little impact on overall assay signal variation 3 For most reaction steps in the assay sequence except for sample standard loading step the biomolecules are present in vast abundance and the binding reactions are completed extremely quickly To ensure good precision it IS recommended that the sample standard incubation should be 20 min 4 Finally the incubation interval when there is no liquid left in the well smooths out the effect of flow rate variances How does the variance CV of Optimiser microplates compare to conventional plates In most assays conventional plates raw signal variance for triplicates is lt 10 which is also true for Optimiser microplates Please see Siloa
15. non conforming product during the warranty period is limited to replacement of or refund for the non conforming product Table of Content SECTION I Using the Optimiser Microplate System tnnt 2 INTRODUCTION RS ne ae tete shies crea ne tete E ete de ee ce 2 MATERIALS PROVIDED AND REQUIRED 5e n interet te ae net tee te iene ae 3 UNIQUE CONSIDERATIONS FOR OPTIMISER MICROPLATE esceseseesseeeneceeaeeeeseeeeaeeeaaesesaeeeseeseaeesaaeeeaaeseaaeseaeeseaeeeeaeees 5 Optimiser Microplate and Assembly cccccsessccccesssssneeeceessnaueeecessseseeeesceeueaeeeescsssseeseesesecsseesesseseueeesessesnseeeeseeeaea 5 Optimiser Microplate Pipetting Instruction iii 5 Avoiding Bubbles While Pipetting ccccccccccsssssssccceessssuececesssseseeeecesssaeseeeecesesaaaeeeesesessaeeessesesusaeeeeseseseeeeeeessesaeess 5 Accurate and Precise Delivery of 5 UL Volumes iii 6 Additional Technical Considerations iii 6 Using Electronic Multi channel Pipette ccce ni aa eiaa ia aaia aaa aaaeaii 6 READER SETUP ciiise E E E A E A e Eaa AEAEE EEE eaa Taa N 8 TUTORIAL 1 PIPETTING TO THE OPTIMISER MICROPLATE inner 10 TUTORIAL 2 IL 6 DEMONSTRATION ASSAY ON THE OPTIMISER MICROPLATE sccecececcesceeseeeaeeeeeeseteneeeeeseeeeaeeeaeens 13 SECTION Il Assay Transfer Guide ire eich icuessodeasecuedihe dueasanevdeseuussncuvancteesuceseereiturciterssqeeuvane 18 SANDWICH ELISA ASSA
16. the capillary barrier is broken and the liquid within the microchannel is drawn out by the absorbent pad and replaced by the new reagent All assay reactions occur within the microfluidic reaction chamber Siloam Biosciences Optimiser Microplate Adoption Kit is designed to provide first time users with a comprehensive introduction to the methods of use and the capabilities of the Optimiser platform Specifically e The Pipetting Instruction Section and Tutorial 1 are designed to guide users through the correct method for pipetting to the Optimiser microplate Although very similar to the conventional 96 well ELISA plate pipetting to the Optimiser requires careful attention to a few key details for reliable performance e Tutorial 2 is designed to allow users to complete a model IL 6 assay Tutorial 2 illustrates that the workflow for Optimiser based assays is similar but much simplified when compared to conventional 96 well ELISA plates by eliminating the traditional wash step Tutorial 2 also shows the capabilities of the Optimiser to deliver equivalent sensitivity to conventional 96 well ELISA plates while using only 5 uL sample volume e The Optimiser Assay Transfer Guide contains detailed instructions for users to transfer a working assay from the conventional 96 well ELISA plate to the revolutionary Optimiser platform Following the step by step instructions users can also successfully test their own assay on the Optimi
17. with appropriate software is recommended Typical Data The IL 6 standard curve ranges from 4 1 to 3000 pg mL Concentration x axis and signal y axis are plotted on Log scales A typical standard curve is presented below Note again that tripling dilutions are used for a wider dynamic range than is typically run in such assays 100000 IL 6 pg mL Average Blank Subtracted gt 3000 36665 36506 10000 1000 31722 31564 amp 333 3 15764 15606 T 111 1 6254 6096 1000 37 0 2572 2414 12 3 1041 883 100 4 1 514 356 1 10 100 1000 10000 0 158 IL 6 pg mL Figure 6 IL 6 Standard Curve with Tabulated Data Note The IL 6 assay provided with the adoption kit is only designed for first time users to verify their methods to run Optimiser based assays The LoQ of this assay lot to lot variations is typically between 4 1 pg mL 37 0 pg mL SIGNIFICANCE OF ASSAY BACKGROUND The reader setup in this example sets a value of 11 000 RFU as the high value for the 50 50 1 saturated substrate signal Regardless of the substrate ratio used the background RFU readings blank signal should not exceed 350 RFU 3 of max value in reader setup e Background signals higher than 3 of RFUmax established during reader setup indicate that one or more steps of the assay was performed incorrectly and users should repeat the assay e Background signals higher than 6 of RFUmax established durin
18. 5 minute incubation steps except for sample which should be at least 20 minutes The Application note section Technical Support Tab of Siloam s website has an article that describes this in greater detail e We recommend that you start with at least 10 minute incubation cycles step but you can certainly use longer up to 20 minutes incubation steps This may be useful when you are processing multiple Optimiser microplates in parallel e All incubation steps must be at least 5 minutes at least 20 minute for sample Incubation steps should not exceed 30 minutes Can I use cell lysate supernatants or other biological fluids such as serum or urine e The flow does work in some circumstances even with particulates in the solution However we have seen that the flow is not very repeatable For these fluids we recommend using supernatant after centrifuging at 13 000 g for 10 minutes or pass through 0 2 um filter How can improve sensitivity of my assay using Optimiser microplate e In most cases using the assay optimization protocol described in the Assay Transfer Guide you should be able to achieve slightly better sensitivity e A guaranteed method to significantly increase sensitivity is the use of repeat load process for sample standard steps Please see the Application Notes under the Support Tab on Siloam s website authored by Tecan BioTek that describe the use of automation stations to increase assay sensitivity mor
19. INSTRUCTION MANUAL Optimiser Microplate Adoption Kit PTIMISER THE NEXT GENERATION OF MICROPLATES SILOAM Better Immunoassays through biosciences we 3 Innovative Microfluidics INTENDED USE The Optimiser Microplate Adoption Kit OPV IL6WDR and the associated Instruction Manual are specifically designed for first time user to provide a comprehensive overview to the Optimiser microplate system Section serves as an Introduction to the Optimiser microplate system and guides the user through correct pipetting techniques with the Optimiser The pipetting technique instructions are accompanied by a Tutorial that will allow users to evaluate their pipetting proficiency on the Optimiser microplate Section also includes detailed instructions for an illustrative IL 6 assay all assay reagents and buffers included with OPV ILGWDR Completing the associated Tutorial will allow users to learn the assay operation sequence for Optimiser microplate assays Successful completion of this assay will also help users understand the POWER OF MICROFLUIDICS to deliver high sensitivity ELISA results with only 5 uL sample volume and a 2 hour assay protocol Section Il is a detailed method description to be used for migrating a validated assay from conventional 96 well plates to Optimiser microplate Section II is presented as a series of 3 experiments where each experiment sequence is described in complete detail includi
20. SERVATIONS AND CONCLUSIONS walt for 10min In each step all wells should be empty within 10 minutes If a well is not empty after 10 minutes please inspect under low power microscope and most likely a bubble will be evident near microchannel interface with well This bubble was accidentally e a eee injected due to incorrect pipetting technique Please refer to the pipetting guidelines and try again 5 ul Red dye e Note that as the dye reagent is changed in the well even a 5 uL volume will clear the seater previous reagent in the microchannel This demonstrates the efficiency of the flushing action instead of the traditional wash step NO EFFECT ON ASSAY PERFORMANCE d a 7 e Observe the wells as they drain out Note the variation in time to empty each well RARE E Bear in mind that as long as each well drains out in 10 minutes this variation has wait for 10 min gt Most assay protocols on Optimiser microplate recommend a 10 minute 5 ul Red dye incubation interval The 20 minute incubation step with red dye shows that ALL wait for 20 min incubations can be extended up to 20 minutes This may be useful for processing J multiple Optimiser microplates in parallel 30 ul OptiWash gt Incubation steps should be at least 5 minutes and no more than 30 minutes Use sus at least 20 minutes incubation for sample standard wait for 10 min Page 10 of 35 Assemble a new Opitmiser microplate 2 and pad on th
21. Technical Assistance If you require assistance please contact Siloam Biosciences Inc Technical Support at 513 429 2976 or techsupport siloambio com Additional technical assistance is available under the Technical Support tab on the Siloam Biosciences web site http siloambio com e Using Optimiser Immunoassay Microplate Video e Optimiser Microplate User s Guide e Reader Settings e Quick Reference Guide e Frequently Asked Questions e Application Notes Two additional videos appear under the Technology tab of the web site e Optimiser Principles of Operation e Running an Assay with Optimiser Dina Siloam Biosciences Inc 413 Northland Blvd Cincinnati OH 45240 USA Phone 1 513 429 2976 Fax 1 513 429 2946 Better Immunoassays Through Innovative Microfluidics www siloambio com biosciences DOC ID OPTI 2 MS 0042 C2 Page 34 of 35 Siloam Biosciences Inc Better Immunoassays Through Innovative Microfluidics SILOAM BIOSCIENCES INC 413 Northland Blvd Cincinnati OH 45240 USA Tel 1 513 429 2976 Fax 1 513 429 2946 http www siloambio com Support techsupport siloambio com
22. The procedure requires total of 75 uL of OptiWash for each assay well to be used Prepare 10 mL of OptiWash into a v shaped reagent reservoir and use it for all wash steps in the assay Page 27 of 35 Assay Layout Capture antibody concentration 4 times as 2 times as conventional assay conventional assay Same as Detection antibody conventional assay concentration dns A Protein Blank Protein Kink Protein Blank lis COMMON tO See conventional assay B standard standard standard slight differences in the c time required for 2 times as ahaa Blank hse Blank oe Blank different wells to empty conventional assay D stangar stancar stangar This difference has no s E Protein Protein Protein Heer EE on asse ame as Blank Blank Blank performance conventional assay F Standard standard standard Procedure 1 10 11 12 13 14 Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page 5 Hint Optimiser incubation steps are from 10 to 20 minutes in length To achieve optimal assay performance all materials must be transferred to the Optimiser microplate within one minute at each step To accomplish this first place the materials to be transferred in the enclosed 96 well polypropylene v bottom plate or v shape reagent reservoir as instructed in Reagent Preparation page 27 Then transfer the materials to the Optimiser well
23. Y TRANSFER GUIDE eseecceeeesceeceeeceesceeseesaeeseecseeeceeaeseaeeaecesecsaesseesaaeeaeeeaeesaeeaeesaesseeseeeneseaeeeaees 19 Requirements for Conventional Assay to be Transferred to Optimiser Microplate 20 HRP Concentrations in Assays on Optimiser Microplate ss 21 Experiment 1 Selecting the Best Antibody Coating Buffer 0 ccccccccccssssececeesssssseceessesseeeecessesssseceeesssssueeeseessaaaes 23 Experiment 2 Selecting the Optimal Capture detection Antibody Concentration 27 Experiment 3 Determine Assay Measurable Range 31 ADVANCED ASSAY PROTOCOL ON THE OPTIMISER MICROPLATE inner 31 OTHER ASSAY FORMATS ON THE OPTIMISER MICROPLATE sesecessceecececeesceeaeeeaecseeceeeseaeeaeeeaeeaaesaeesaeeseeseaseaaeeaeeaee 31 TROUBLESHOOTING issii a ei E a A E aed dla dead oe sede nette a eee a tirs 32 APPENDIX 1 ULTRA HIGH SENSITIVITY ASSAYS ON OPTIMISER MICROPLATE ie 33 A Symbol indicates mandatory step required to ensure proper operation Q Symbol indicates helpful tips to achieve optimal performance SECTION I Using the Optimiser Microplate System INTRODUCTION Siloam Biosciences Optimiser technology platform offers a rapid and sensitive chemifluorescent based ELISA procedure that uses very small sample volumes The speed sensitivity and small sample requirements are enabled by the unique microfluidic design of t
24. at same procedure till well H1 then continue to well B2 and repeat till well G2 leaving well H2 as zero point blank 1 2 A _no dilution 1 256 B 1 2 1 512 C 1 4 1 1024 D 1 8 1 2048 E 1 16 1 4096 F 1 32 1 8192 G 1 64 1 16384 LH 1 128 Zero OptiWash onl Page 8 of 35 Transfer to Optimiser microplate and read Transfer 4uL solution of each well in the V bottom plate to the corresponding well on column 1 and 2 of Optimiser microplate Wait for all the wells to empty Read the Optimiser microplate with fluorescence reader In order to read Optimiser microplate adjust your reader setting as listed in page 8 to enable the following requirements e For the capacity to run a wide dynamic range assay e g 729 fold the reader should have o Detectable dose response from well A1 to D2 e g 39747 vs 95 RFU AND o Clearly distinguish well D2 to well H2 e g 95 vs 22 RFU e For the capacity to run a more limited dynamic range assay 64 fold the reader should have o Detectable dose response from well C1 to D2 e g 17735 vs 95 RFU AND o Clearly distinguish well D2 to well H2 e g 95 vs 22 RFU When using same reader setting to read IL 6 demo assay on the Optimiser microplate the top signal should be close to value of well A1 and the background signal should be close to value of well B2 A2 Record the value of RFUmax from A1 which will be used as reference in assay transfer proc
25. bation read the plate If all the assay reagent preparation steps and protocol are followed correctly the wells microfluidic channels corresponding to top 2 up to top 3 standards clearly appear pink owing to developed substrate If the pink color is not evident even for topmost standard one or more reagent preparation steps or assay steps was not performed correctly Page 16 of 35 It is common to see slight differences in the time required for different wells to empty This difference has no impact on assay performance To facilitate work flow incubations designated as 10 minutes may be extended to 20 minutes with no impact on method performance Optimiser washes are performed by simply dispensing OptiWash to the wells Wipe the plate bottom thoroughly Any liquid residue on the bottom surface will cause false positive signal In rare cases lt 0 2 a well may not empty in 10 min If so blot the reagent from the well with a tissue Do not include data from this well in calculations Calculations 1 Calculate the mean background signal from the blank wells wells containing OptiBlock only at the sample incubation step 2 Subtract the mean background signal from the signal of individual standard 3 Create a standard curve by plotting the standard concentration x axis vs the background adjusted signal in relative fluorescence units RFU y axis A five parameter logistic curve fit
26. concentration curve ONLY if the peak RFU value is less than 90 of RFU max Use the LOW concentration curve ONLY if the peak RFU value is more than 10 of RFU max If both curves are valid per criteria listed above usually the same OptiBind formulation will show best results If there is discrepancy in choice of OptiBind formulation from LOW and HIGH concentration curves use the HIGH concentration curve to make the selection Example Data Figure 9 shows screening test results for the illustrative IL 6 assay Protein standard concentrations of 200 pg mL 80 of max and 50 pg mL 20 of max were used 2 ug mL of capture antibody and 2 ug mL of detection antibody were used for this assay Data read using Biotek FLx800 fluorescence plate reader with excitation filter at 528 20nm and emission filter at 590 35 sensitivity at 45 Data acquisition and analysis utilized Gen5 software and Excel ove Coating buffer screening for Illustrative IL 6 assay 50 pg ml IL 6 6000 E 200 pg mL IL 6 5000 4000 3000 2000 RFU background subtracted 1000 A B C D E F G H l J K L OptiBind Type A L Figure 9 Results for coat buffer screening test for Illustrative IL 6 assay For the data shown in Figure 9 both curves for screening test result are valid and either curve can be used to select the best OptiBind buffer formulation Both curves also demonstrate that OptiBind H is the best coat buffer for this assay Page 25
27. dard for experiment 3 is Tested By Date Page 30 of 35 Experiment 3 Determine Assay Measurable Range The coating buffer selected from Experiment 1 and antibody concentrations selected from Experiment 2 will be used for the final Experiment to determine the dynamic range of the assay This experiment will run a standard curve of the assay with a wide range of concentrations which covers the expected dynamic range Most Optimiser microplate based assays are expected to have a dynamic range of 730 fold 1 3 dilution 7 concentrations Appropriate standard diluents must be used for this experiment For example use cell culture medium as standard diluent for measuring cell culture supernatant it is important to match the matrix of your intended sample ADVANCED ASSAY PROTOCOL ON THE OPTIMISER MICROPLATE Unique Ultra High Sensitivity Repeat Loading Protocol As the Optimiser microplate uses a flow through principle where subsequent reagent analyte additions flush microchannel contents onto the absorbent pad multiple analyte additions can be used to increase sensitivity Please see detail description in Appendix and example assays in Application Notes on Siloam s website Ultra Fast Sandwich ELISA Protocol The total assay time for a standard Optimiser based assay is less than 2 hours which already represents a significant time saving and increase in throughput However based on kinetics study even 5 min of incubation
28. e 23 of 35 Assay Layout OptiBind Type A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 A High concentration protein standard concentration at 80 of top B standard used in conventional assay C D Low concentration protein standard concentration at 20 of top E standard used in conventional assay F G Zero blank H Procedure 1 Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page 5 2 Hint Optimiser incubation steps are from 10 to 20 minutes in length To achieve optimal assay performance all materials must be transferred to the Optimiser microplate within one minute at each step To accomplish this first place the materials to be transferred in the enclosed 96 well polypropylene v bottom plate or v shape reagent reservoir as instructed in Reagent Preparation page 23 Then transfer the materials to the Optimiser wells using a 10 uL multi channel pipette 3 Dispense 5 uL capture antibody solution to the required number of wells in the Optimiser microplate Incubate 10 minutes at room temperature RT 4 Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step 5 Dispense 5 uL OptiBlock to the capture antibody coated wells Incubate 10 minutes at RT 6 Dispense 5 uL of the standard and blank to the required number of replicate wells of the plate Incubate
29. e holder Repeat the dispensing protocol shown above again in ALL 12 Columns of Optimiser microplate 2 Time the dispensing cycles and check that all dispensing steps are completed within 1 minute CHECK THAT ALL WELLS DRAIN WITHIN 10 MINUTES FOR EACH DISPENSING STEP If any wells take longer than 10 minutes the most likely cause is an error in pipetting causing a visually evident or micro bubble Please refer to the pipetting instructions and repeat steps 6 and 7 User MUST complete dispensing protocol on to entire Optimiser microplate with all wells on that microplate draining in 10 minutes for all steps in protocol Optimiser microplate is a powerful tool for ELISAs and requires correct pipetting procedures to ensure repeatable results USERS MUST BE ABLE TO COMPLETE_THE DISPENSING PROTOCOLS FOR A COMPLETE Optimiser MICROPLATE WITH ALL WELLS DRAINING IN LESS THAN 10 MINUTES FOR ALL STEPS OF THE PROTOCOL THIS SIMPLE STEP IS CRITICAL TO ENSURE USERS CAN ACHIEVE EXCELLENT ASSAY RESULTS ON OPTIMISER MICROPLATE Two additional Optimiser microplates are included with the package to allow users to further practice and perfect pipetting to the Optimiser microplate Excess volumes of the red green dye and OptiWash are also included OPTIONAL Procedure with Electronic Multi channel Pipette 5 100 uL volume 1 2 3 Repeat the protocol described for Manual multi channel pipette with an Electronic
30. e than 100x Please discuss your application with Siloam s tech support and we can offer more accurate guidance Page 22 of 35 Experiment 1 Selecting the Best Antibody Coating Buffer In Optimiser microplate all assay reactions occur in the microfluidic microchannel The high surface area to volume ratio and short diffusion distances of the microchannels allow rapid protein adsorption onto the surface Unlike the assay in conventional plate the capture antibody adsorption in Optimiser microplate is dominated by the reaction rate of protein adsorption which is strongly affected by the coating buffer The first step of assay development is to screen twelve types of OptiBind coating buffer provided in the kit It requires one assay experiment and uses one full Optimiser microplate The assay sensitivity can vary as much as 10x depending on the coat buffer used for capture antibody coating This additional assay optimization step is critical for Optimiser microplates to achieve best performance OptiBind COATING BUFFER IS MANDATORY FOR OPTIMISER ASSAYS DO NOT USE ANY OTHER COATIONG BUFFER Reagent Preparation The incubation times for Optimiser are only 10 20 minutes Preparing all the reagents samples standards in advance will allow for proper timing especially for first time users Always prepare extra volume of solution for easy transferring Prepare 30 uL extra volume in each well of 96 well v bottom plate
31. ecisely delivering 5 uL Multichannel pipette capable of delivery of 30 uL Vortex mixer Fluorescence plate reader and control software Analytical software Microcentrifuge Timer Do gI UT 0 RS Page 4 of 35 UNIQUE CONSIDERATIONS FOR OPTIMISER MICROPLATE Optimiser Microplate and Assembly Optimiser Microplate Optimiser Pad i imi lt Optimiser Pa Figure 2 Optimiser microplate assembly Position absorbent pad on holder align the Optimiser yo Holder microplate and press down gently to click lock the plate in holder Optimiser Microplate Pipetting Instruction Tutorial 1 provides hands on training for first time users to practice pipetting with Optimiser microplate Please read the entire Pipetting Instruction section before attempting Tutorial 1 Avoiding Bubbles While Pipetting 1 Bubbles will compromise the performance of assays on Optimiser microplate by interfering with the flow of liquid within the microchannels 2 OptiBlock reagent may form bubbles readily with standard pipetting techniques 3 To avoid complications due to bubbles Siloam Biosciences recommends the use of the Reverse Pipetting technique during all pipetting steps a To aspirate liquid press the operating button of the pipette to the second stop refer to illustration below b Immerse the pipette tip in the liquid to a depth of about 2 mm and steadily release the operating button completely c
32. ed by simply dispensing OptiWash to the wells Caution In rare cases a well may not empty in 10 min If so blot the reagent from the well with a tissue Do not analyze signal from this well Calculations 1 Calculate the mean background signal from the blank wells for assay with each type of OptiBind coating buffer for example average signal from well G1 and H1 for background signal of assay with OptiBind A coating buffer Calculate the mean sample signal from the high concentration protein standard wells for assay with each type of OptiBind coating buffer for example average signal from well A1 B1 C1 for high signal of assay with OptiBind A coating buffer Calculate the mean sample signal from the low concentration protein standard wells for assay with each type of OptiBind coating buffer for example average signal from well D1 E1 F1 for low signal of assay with OptiBind A coating buffer Create screening curves by plotting the OptiBind coating buffer types x axis vs the background adjusted signal y axis Choose the type of OptiBind coating buffer which gives highest signal after subtracting background See Figure 9 Selecting the Best Coating Buffer Select the OptiBind buffer type which yields the maximum signal This particular OptiBind coating buffer should be used for all further experiments of this assay i e using this particular capture antibody clone Use the HIGH
33. edure Note please transform your reading from RFU relative fluorescence unit to percentage of RFUmax from well A1 for comparing your data to the typical data provided below The following results show data on a BioTek FLx800 Fluorescence Plate Reader with excitation filter at 529 20 emission filter at 590 35 and sensitivity at 45 Dilution from 5x Percentaze Well fully developed RFU of Max substrate Al Desired range for e ais reader B1 2 28662 72 11 Detectable dose C1 4 17735 44 62 response should D1 8 9943 25 01 RL E1 16 5225 13 15 dilutions less than aa T 3 oe zoey Res F1 2776 6 98 background G1 64 1443 3 63 H1 128 771 1 94 Typical assay background A2 256 458 1 15 B2 512 266 0 67 C2 1024 162 0 41 D2 2048 95 0 24 Clear difference in E2 4096 62 0 15 signal intensity F2 8192 41 0 10 perwecn wel Dz G2 16384 32 0 08 and H2 H2 OptiWash blank 22 0 06 Page 9 of 35 TUTORIAL 1 PIPETTING TO THE OPTIMISER MICROPLATE Materials Required for Pipetting Tutorial and Supplied with Optimiser Microplate Adoption Kit 1 One Optimiser holder 2 Same Optimiser microplate 1 used for reader setup 3 or more unused columns will be used for pipetting 3 One new Optimiser microplate 2 all 12 columns will be used for pipetting 4 One Optimiser pad single use 5 OptiWash buffer 6 Green dye solution 6 Red dye solut
34. efore plate is read Unexpectedly high signal Incorrect reader filters with overlapped wavelength bandwidth Confirm filters meet requirements for substrate Reagent contamination Avoid cross contamination in reagents Always change the pipette tips when handling different buffers reagents Poor precision Pipetting errors use of alternate assay buffers or SAv HRP Follow recommendations for pipetting small volumes Page 6 Variance lt 10 and background lt 3 of RFUmax established during reader setup are expected Do not substitute provided assay buffers or SAv HRP Signal of lower standard s are lt 0 following Degraded standard Use standard on the day of its reconstitution or Thaw single use aliquots fresh on each test day Avoid repeated freeze thaws background subtraction Degraded capture antibody Use within specified expiration period Store according to recommended storage temperature Page 32 of 35 APPENDIX 1 ULTRA HIGH SENSITIVITY ASSAYS ON OPTIMISER MICROPLATES Because of the unique features of the Optimiser plate and OptiMax ELISA procedures users can apply sample to individual microfluidic reaction chambers multiple times The result is a significant improvement in assay sensitivity when ultra high sensitivity is required The additional sample applications can be performed manually for a limited number of repeat sample loads but Siloam strongl
35. ely with 40 uL OptiBlock blocking buffer Vortex the 3000 pg mL standard briefly to mix c Standard Curve Prepare the remaining IL 6 standards by performing six serial three fold dilutions in OptiBlock beginning with the 3000 pg mL standard as follows i Dispense 120 uL of Standard 1 3000 pg mL to well A1 of the 96 well polypropylene v bottom plate ii Dispense 80 uL OptiBlock to each of the seven wells of the same column immediately below the 3000 pg mL containing well wells B1 H1 iii Transfer 40 uL of the 3000 pg mL standard from well A1 to well B1 immediately below it Mix the contents of well B1 gently Then transfer 40 uL from well B1 to well C1 change tips and titrate iv Continue serial dilutions while changing tips after each 40 uL transfer and before mixing until the 4 1 pg mL standard has been created in the seventh well well G1 of the column v Do not transfer IL 6 solution to the eighth well H1 It contains OptiBlock only and will provide material for the blank wells Page 14 of 35 1 1 A 120 uL Std 1 A 3000 B 80 uL Blocking B 1000 C 80 uL Blocking C 333 3 D 80 uL Blocking D 111 1 E 80 uL Blocking E 37 0 F 80 uL Blocking F 12 3 G 80 uL Blocking G 4 1 H 80 uL Blocking H 0 5 Detection Antibody The procedure requires 5 uL of the detection antibody working solution for each assay well to be used a Prepare a 1 25 dilution of the
36. equired number of wells in the Optimiser microplate Incubate 10 minutes at room temperature RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL OptiBlock to the capture antibody coated wells Incubate 10 minutes at RT Dispense 5 uL of the standard and blank to the required number of replicate wells of the plate Incubate 20 minutes at RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL detection antibody working solution to each well Incubate 10 minutes at RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL SAv HRP to each well Incubate 10 minutes at RT Dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Again dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL OptiGlow working solution to each well Incubate for 15 minutes at RT a Caution Observe the wells during the incubation When the substrate has completely drained from all wells remove the plate and pad from the holder Discard the pad Wipe the bottom of the plate with a Kimwipe to remove any liquid on the bottom surface of the plate Step 13a will be completed within the 15 minute substrate incubation time Place the plate in the reading chamber of a fluorescence plate reader Promptly at the conclusion of the 15 minute incu
37. g reader setup corresponding to 700 RFU in current example indicate a failure and the assay must be repeated CAUSES e High backgrounds are most commonly a result of pipetting errors and can be resolved with careful attention to the procedure and additional practice e Another common cause for high background is use of alternate SAv HRP or direct HRP labeled detection antibodies Optimiser based assays are exquisitely sensitive to HRP concentration and the SAv HRP provided by Siloam has been carefully optimized to achieve best performance Use of alternate buffers especially blocking buffers can also lead to high background signals Please consult with Siloam s Tech Support team before substituting any of the buffers provided with the kits or as part of the OptiMax assay buffer reagent sets TUTORIAL 2 TARGETED OUTCOME First time users can run a complete assay on the Optimiser microplate and confirm that they can generate similar data as listed in the User Manual This Tutorial is intended to serve two purposes a to familiarize users with the assay operation sequence on the Optimiser microplate and ensure performance matches with Siloam s data and b to provide users an introduction to the capabilities of the Optimiser microplate to deliver high sensitivity assay data even when using only 5 ul sample volumes and a 2 hour assay protocol Assay Background Page 17 of 35 SECTION II Assay Transfer Guide
38. h a single channel pipette A multichannel pipette is essential to ensure that all dispense steps can be comfortably completed in 1 minute or less With a single channel pipette it is very difficult to complete pipetting to even 3 columns in 1 minute How critical is the accuracy of 5 pl dispensing volume The Optimiser microplate is designed such that the 5 ul volume represents a slight excess compared to the microchannel internal volume Provided that the dispense volume is greater than 4 5 ul slight even up to 10 dispense volume variations will not affect assay results Why has the recommended operating volume been changed to 5 uL remember seeing 10 pL as recommended volume in earlier version of the FAQ 1 Minimizing the volume helps with improving the precision When using the 10 ul protocol there is higher variation in the time to empty for different wells on each plate This is related to the flow rate of the microchannel and larger volume show more net effect on flow duration and variation of the duration 2 The new 5 ul protocol also reduces the incidences of slow or stopped flow With proper pipetting technique and by use of the new protocol our lab tests show that flow failure rate well does not empty after 10 minutes is now less than 0 2 3 We have verified through extensive assay tests that change from 10 ul to 5 ul does not affect the assay sensitivity This is partly owing to improvements made t
39. he Optimiser microplate Standard immunoassay reactions such as analyte capture and detection occur within a 5 wl microfluidic reaction chamber The unique microchannel geometry and small reaction volumes favor rapid reaction kinetics Typical Optimiser assay procedures utilize 5 uL sample volumes and each reaction step is completed within 50 10 minutes Most standard Optimiser technology based ELISAs are completed within approximately 2 hours Please refer to the Optimiser Technology page on Siloam s website www siloambio com for more details regarding the principles behind the Optimiser microplate platform Figure 1 Optimiser microplate The Optimiser microplate is a revolutionary new microplate format With an ANSI SBS compliant 96 well layout the Optimiser microplate integrates the Power of Microfluidics to allow for low volume rapid and uniquely high sensitivity immunoassay protocols Figure 1 shows the Optimiser microplate schematic with a magnified view of one cell of the Optimiser microplate Each cell of the Optimiser microplate has a loading well only used to add reagents and a microfluidic reaction chamber Reagents samples are added to the well and transported via capillary action to an absorbent pad not shown The unique design of the Optimiser microplate allows the well to be drained but each liquid is trapped in the channel by capillary forces As the next liquid volume is added
40. ion Other Materials Equipments Required 1 Kimwipes or other laboratory tissue paper 2 Reagent reservoirs V shape reservoir 3 Single channel pipette capable of delivering in the ranges of 100 1000 uL 4 Pipette tips for delivering in the ranges of 100 1000 uL 5 Multichannel pipette capable of accurately and precisely delivering 5 uL 6 Multichannel pipette capable of delivery of 30 uL Procedure with Manual Multi channel Pipette 1 Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page 5 2 Transfer 1 5 mL of green dye solution into a V shape reagent reservoir Transfer 1 5 mL of red dye solution into another V shape reagent reservoir Transfer 3 mL of OptiWash solution into a V shape reagent reservoir 3 To aspirate liquid hold the multi channel pipette nearly vertical and immerse the pipette tip in the liquid to a depth of approximately 2 mm in the liquid Withdraw the operating button steadily Wait 1 second Withdraw the tip from the liquid 4 Dispense into columns 2 4 3 columns for Optimiser microplate 1 per the sequence illustrated below a To dispense liquid hold the multi channel pipette nearly vertical With ALL the pipette tips touching the surface of the Optimiser wells depress the operating button steadily until the liquid is dispensed DO NOT position pipette tips into the hole at the bottom of the surface 5 ul Green dye OB
41. ion of 20 of top standard diluted in OptiBlock in a v shape reservoir 250 pg mL of IL 6 is the top standard in the example assay Hence 50 pg mL IL 6 standard would be prepared for this step 6 Blank zero Prepare 1 mL of OptiBlock in a v shape reservoir 7 Detection Antibody Use same concentration as conventional assay prepare the detection antibody working solution by diluting the detection antibody stock in OptiBlock to make 1mL final working solution in a v shape reservoir 2 pg mL detection antibody concentration is used in the example assay Hence 1 mL of 2 ug mL detection antibody working solution in OptiBlock would be prepared for this step 8 SAv HRP Use SAv HRP stock solution provided in the kit Prepare the SAv HRP working solution by adding 8 uL of SAv HRP stock solution to 1 2 mL of OptiBlock 1 150 dilution in a v shape reservoir mix well 9 Substrate solution The procedure requires 10 uL of the working substrate solution for each assay well to be used Prepare the substrate working solution in a v shape reagent reservoir by mixing 0 9 mL of OptiGlow A 0 9 mL of OptiGlow B and 18 uL of OptiGlow C 10 OptiWash OptiWash is provided in ready to use form No further preparation is required The procedure requires total of 75 uL of OptiWash for each assay well to be used Prepare 10 mL of OptiWash into a v shaped reagent reservoir and use it for all wash steps in the assay Pag
42. l of a single column in the polypropylene 96 well v bottom plate 8 OptiWash OptiWash is provided in ready to use form No further preparation is required The procedure requires 75 uL of OptiWash for each assay well to be used Dispense 4 mL OptiWash buffer to a v shaped reagent reservoir and use for all wash steps in the assay DO NOT SUBSTITUTE OTHER BUFFERS OR REAGENTS FOR THOSE PROVIDED WITH THE KIT OptiMax buffers are specially formulated to work with the Optimiser microplate and substitute buffers or reagents may lead to poor assay performance ENSURE THAT TIP CHANGES AS RECOMMENDED FOR STANDARD PREPARATION ARE FOLLOWED Continued use of same tip may lead to errors in dilution and consequent assay signals Page 15 of 35 Procedure 1 10 11 12 13 14 Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page 5 Hint Optimiser incubation steps are from 10 to 20 minutes in length To achieve optimal assay performance all materials must be transferred to the Optimiser microplate within one minute at each step To accomplish this first place the materials to be transferred in the enclosed 96 well polypropylene v bottom plate or v shape reagent reservoir instructed in Reagent Preparation page 14 Then transfer the materials to the Optimiser wells using a multi channel pipette Dispense 5 uL capture antibody working solution to the r
43. labeled with HRP a biotin labeled detection antibody is most preferred see next page for details e Known dynamic range for assay is used as a starting point for the assay transfer process IT IS STRONGLY RECEOMMENDED THAT ALL ASSAY MATERIALS USED SHOULD BE TESTED IN COLORIMETRIC FORMAT RIGHT BEFORE THE ASSAY MIGRATION PROCESS TO ENSURE MATERIAL QUALITY DO NOT USE ELISA REAGENT MANUFACTURER S SPECIFICATIONS WITHOUT A CONFIRMATORY EXPERIMENT VERIFYING PERFORMANCE IN COLORIMETRIC MODE As an example a working IL 6 colorimetric assay with conventional 96 well plate is shown below The assay transfer guide uses this assay as an example to illustrate the transfer process 10 000 conventional 96 well colorimetic IL 6 pg mL Average OD450 3 Blank Subtracted g 250 1 586 1 561 51 000 125 0 823 0 798 63 0 441 0 416 Ga 31 0 247 0 222 80 100 16 0 145 0 120 D 0 089 0 064 a 0 059 0 034 0 010 pee et ae 0 0 025 1 10 100 1000 Human IL 6 pg mL Figure 8 Standard curve of IL 6 assay run in conventional 96 well plate using TMB substrate and colorimetric detection of absorbance at 450 nm and corrected with 630 nm 2 ug mL concentration for Capture and Detection antibody Details for illustrative IL 6 assay e Assay metrics o Background OD 0 025 less than 0 15 e g after a standardized colorimetric substrate development time such as 15 minutes o Max signal OD 1 58 more than 1
44. lel ipetting P Frequently Asked Questions Almost all pipetting protocols specify users NOT to touch the well surface during pipetting Why does the Optimiser microplate user guide suggest the exact opposite In conventional 96 well ELISA plates if the pipette tip touches the bottom surface of the well it may physically disrupt some of the bound bio molecules In the Optimiser microplate all the assay reactions occur within the microchannel Hence touching the pipette tip on the loading well of the Optimiser has absolutely no effect on the assay performance For most dispensing steps in Optimiser based assays users are dispensing only 5 ul volumes If the pipette tip does NOT touch the well surface the dispensed well volume may bead and stick to the end of the tip The well geometry of the Optimiser is engineered to ensure smooth filling of well microchannel provided the liquid is dispensed steadily and directly on the well surface See the Optimiser Technology page on Siloam s website for instructional videos on pipetting techniques Why must all materials be transferred to the Optimiser microplate within one minute at each step in the assay procedure Optimiser incubation steps are from 10 to 20 minutes in length Longer time to transfer material will cause time difference between each well in incubation which may affect the assay accuracy don t have a multichannel pipette can I try the kit wit
45. m s website for a Technical Note detailing variance studies on the Optimiser platform For first time users it is common to see variances at 15 and even up to 20 In almost all cases this is related to pipetting techniques and as any other platform practice makes perfect most users see noticeable improvement after running a few Optimiser plates The most common pipetting related issues that are resolved with careful attention to details and practice include 1 Tips do not touch well surface leading to incomplete dispensing This can lead to very high signal e g if block buffer is not properly dispensed or very low signals e g if SAV HRP is not properly dispensed 2 Inconsistent load times as users learn pipetting procedures With significant differences between loading intervals across the plate there is higher variance with practice users can establish a steady rhythm with improved precision 3 Failure to change tips in reagent preparations and or between dispensing steps for assay sequence 4 Use of inappropriate pipette tips and or pipettes Most users after completing 10 12 Optimiser based assays can achieve a background signal lt 2 3 of peak signal see Page 15 and b variance lt 10 for all points on standard curve These can be used as metrics to determine user proficiency on the Optimiser system Page 12 of 35 TUTORIAL 2 IL 6 DEMONSTRATION ASSAY ON THE OPTIMISER MICROPLATE The
46. multi channel pipette Choose Reverse Pipetting in function settings for Electronic pipette Choose Multiple Dispensing mode and program for 12 dispense cycles with 5 ul dispense volume per cycle for dispensing to all 12 columns OBSERVATIONS e All observations and conclusions listed for previous protocol e Note the difference in time required to load 12 columns with an electronic multi channel pipette Page 11 of 35 Variance Frequently Asked Questions If one well drains in say 1 minute and another in say 8 minutes how is it possible that they provide comparable results Although it may seem that difference of minutes may have an impact on the assay precision Siloam has demonstrated with multiple assays that well optimized assays on Optimiser microplate easily achieve CV lt 6 10 The minimal effect of flow rate on precision is a combination of multiple factors 1 On the micro scale reaction kinetics are vastly different compared to the macro scale kinetics of conventional 96 well ELISA plate In microfluidic channels most surface binding reactions are saturated in 5 minutes Optimiser characterization data shows that up to 75 of peak adsorption is completed in only 10 seconds and assay binding reactions saturate in 5 minute This is a result of two factors a The diffusion distances in the microchannel are extremely small the channel has a cross section of only 200 um x 200 um hence diffusion is
47. n Optimiser microplate will be used for this experiment Based on the result from Experiment 1 concentration of protein standard which gives maximum signal between 10 to 50 of RFUmax Will be used for this experiment Reagent Preparation Bring all reagents to room temperature before use and prepare all necessary dilutions before beginning the test procedure 1 2 OptiBind Use the OptiBind coat buffer selected from experiment 1 Capture Antibody Three concentrations of capture antibody working solution will be tested a same b 2 times and c 4 times as that used in conventional assay Prepare the capture antibody working solution by diluting the capture antibody stock in selected OptiBind to make 250 wl final working solution Use a single row in the polypropylene 96 well v bottom plate load 60 pL of working solution to each well Load wells 1 4 with 4x concentration capture antibody solution wells 5 9 with 2x concentration and wells 9 12 with same as conventional concentration OptiBlock OptiBlock is provided in ready to use form and is used to block the surfaces of the Optimiser s microfluidic reaction chambers following their incubation with the capture antibody solution OptiBlock is also used as the diluent for the protein standard detection antibody and SAv HRP For blocking step prepare 1 mL of OptiBlock in a v shape reservoir Protein Standard Prepare 1 mL of protein standard wi
48. ng reagent preparation steps assay plate layouts assay procedures calculations and data analysis methods Section II also describes an illustrative IL 6 assay and the results from the IL 6 assay experiments are used to illustrate the data analysis methods used in the assay transfer guide Instruction Manual Optimiser Microplate Adoption Kit For adapting conventional ELISA plate assays to Optimiser Microplate System Catalogue Numbers OPV IL6WDR Manufactured by Siloam Biosciences Inc 413 Northland Blvd Cincinnati Ohio 45240 USA FOR RESEARCH USE ONLY Not for use in clinical diagnostic procedures Read the Instruction Manual in its entirety before using the Optimiser Microplate Adoption Kit Optimiser microplates are warranted to perform in conformance with published product specifications in effect at the time of sale as set forth in product documentation and or package inserts Products are supplied for Research Use Only The use of this product for any clinical diagnostic applications is expressly prohibited The warranty provided herein is valid only when used by properly trained individuals and is limited to six months from the date of shipment and does not extend to anyone other than the original purchaser No other warranties express or implied are granted including without limitation implied warranties of merchantability fitness for any particular purpose or non infringement Buyers exclusive remedy for
49. nverted to S N ratio Capture antibody concentration Detection antibody 8 pg mL 4 g mL 2 pg mL concentration 1 2 3 4 5 6 7 8 9 10 11 12 8ug m H 8 03 672 4 60 B 4 g mL 8 34 9 00 6 50 D aman E 9 85 7 17 5 28 F e 8 ug mL capture antibody concentration and 2 ug mL detection antibody concentrations are selected for the highest S N ratio Page 29 of 35 Data logging and Calculation Worksheets Experiment 2 Selecting the Optimal Capture detection Antibody Concentrations Assay Target Capture antibody information a and working concentration 1x ug mL 2x __ g mL 4x _ __ug mL Detection antibody information _ and working concentration 1x ug mL 2x ___ g mL 4x _ __ug mL Protein standard information P Test results Capture Antibody Detection antibody 1 2 3 4 5 6 7 8 9 10 11 12 A 1x B C 2x D E 4x F Calculated Mean Capture Antibody 4 times as conventional assay 2 times as conventional assay Same as conventional assay Detection antibody standard Zero S N Standard Zero S N Standard Zero S N 4 times as A conventional assay 2 times as Same as conventional assay B C conventional assay D E F Optimal capture antibody concentration __ ug mL Optimal detection antibody concentration __ ug mL With optimal antibody concentrations the top concentration of protein stan
50. o further preparation is required Do not substitute other coating buffers for OptiBind H Capture Antibody The procedure requires 5 uL of capture antibody working solution for each assay well to be used a Prepare a 1 62 5 dilution of the capture antibody stock in OptiBind H buffer in a clean polypropylene tube such as Eppendorf tube Add 8 ul of capture antibody stock solution to 0 492 mL of OptiBind H buffer b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate OptiBlock OptiBlock buffer is provided in ready to use form and is used to block the surfaces of the Optimiser microplate s microfluidic reaction chambers following their incubation with the capture antibody solution OptiBlock is also used as the diluent for the standard detection antibody and SAv HRP in this experiment Recombinant IL 6 Standard a Stock Solution The IL 6 standard is provided in lyophilized form i Reconstitute the lyophilized standard by adding 420 uL OptiBlock blocking buffer ii Mix by gentle swirling until all of the lyophilized material has dissolved iii Vortex gently to ensure thorough mixing of the reconstituted standard iv Use freshly prepared material on the day of reconstitution b Working Solution The concentration of the reconstituted IL 6 standard is 4 ng mL Prepare a 3000 pg mL standard Standard 1 by mixing 120 uL IL 6 standard appropriat
51. o the OptiMax buffer formulations Page 7 of 35 FLUORESCENCE PLATE READER SETUP Optimiser microplate based assays are compatible with standard fluorescence plate readers and multi mode plate readers with fluorescence read capability Below is the general guidance for setting up the readers The Technical Support section on Siloam s website offers detailed guidance on the set up of several major brands of instruments as illustrative examples Step 1 Selecting the wavelength for excitation and emission light 450 500 570 lt 585 550 600 650 700 Wavelength nm Figure 5 Normalized absorption left an d emission right spectra of OptiGlow chemifluorescent substrate Step 2 Selecting the plate type The Optimiser microplate fits the 96 well SBS standard in all specifications Please use 96 well standard or similar selection when setting the plate type Step 3 Selecting the probe direction Use top reading for probe direction Step 4 Selecting the sensitivity gain When defining reading parameters for fluorescence analysis setting the photomultiplier tube PMT sensitivity referred to as gain in some types of fluorescence readers is important for obtaining useful measurements A manual sensitivity gain setting is recommended for reading Optimiser microplates The procedure is described below Assays performed on the Optimiser platform use the OptiGlow substrate
52. of 35 Data logging and Calculation Worksheets Experiment 1 Selecting the Correct Antibody Coating Buffer Assay Target S OoOo Capture antibody information __ and working concentration __ _ug mL Detection antibody information __ and working concentration __ _ug mL Protein standard information and top concentration _ 2 High protein standard 80 of top standard concentration __ a SN Low protein standard 20 of top standard concentration __ REV o S Test results OptiBind type A B C D E F G H J K L 1 3 4 5 6 7 8 9 10 11 12 A High B standard C Mean D Low E standard F Mean G Blank H Mean OptiBind type _ With this type of OptiBind protein standard at concentration RFU max It will be used for experiment 2 Tested By Date Page 26 of 35 gives highest signal if that signal of high standard between 10 and 90 of RFU max give signal between 10 and 50 of Experiment 2 Selecting the Optimal Capture and Detection Antibody Concentrations The larger surface area and very high surface area to volume ratio in the microfluidic channel of Optimiser microplate allows more capture antibody to be adsorbed onto the surface which may improve assay sensitivity This experiment uses a checkerboard titration pattern with 3 concentrations of capture antibody and 3 concentrations of detection antibody Six rows 72 wells in a
53. on Liquid does not drain from the Optimiser well or does not drain within 10 minutes A bubble is in the well Disrupt the bubble with a clean 26 gauge needle Follow recommended pipetting guidelines Prepare excess reagent to avoid aspirating air Do not use detergents Sample contains particulates Centrifuge sample for 10 min at 13 000 RPM or Filter the sample using a 0 2 um filter Plate has lost contact with the absorbent pad or is positioned incorrectly Ensure that the absorbent side rough of the pad is in contact with Optimiser micropate and the tape side smooth is facing down to touch holder Ensure the topside of the pad is touching the bottom of Optimiser microplate by pushing down firmly on the 4 corners of the plate Ensure the plate and pad are securely aligned in the holder No signal or unexpectedly low signal Standard has degraded Incorrect reader filters Use standard on the day of its reconstitution or Thaw single use aliquots fresh on each test day Avoid repeated freeze thaws Confirm filters meet requirements for substrate Antibodies or SAv HRP are degraded Use within specified expiration period Store according to recommended storage temperature Substrate was prepared Thaw OptiGlow C thoroughly before preparing incorrectly substrate working solution Substrate working solution has e Prepare substrate no more than 30 minutes degraded b
54. roxidase has been captured on the microchannel surface during the sequence of reactions cited above the enzyme will react with the substrate solution and will yield a fluorescent signal when excited at the appropriate wavelength Within the linear portion of the curve the light signal emitted will be directly proportional to the concentration of antigen in standards and samples and will be quantifiable when the plate is read using a fluorescence plate reader In order to achieve best assay performance with Optimiser platform serial optimization tests for each type of assay need to be performed before measuring the real sample A well characterized and robust assay on 96 well platform is a mandatory pre requisite for the Assay Transfer Process The assay transfer is a 3 step process with step 1 being data collection for conventional assay Second run one experiment to screen 12 types of supplied OptiBind coating buffers to determine the best coating buffer to use for this assay Third run a checkerboard titration experiment to determine the concentration of capture antibody and detection antibody which gives the best signal noise ratio Finally run an assay with wide range of target protein concentration with the selected optimal coating buffer and antibodies concentrations to determine the measurable dynamic range of the assay Gathering information about conventional assay Experiment 1 selecting the best antibody coating buffer Experiment 2
55. s using a 10 uL multi channel pipette Dispense 5 uL capture antibody solution to the required number of wells in the Optimiser microplate Incubate 10 minutes at room temperature RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL OptiBlock to the capture antibody coated wells Incubate 10 minutes at RT Dispense 5 uL of the standard and blank to the required number of replicate wells of the plate Incubate 20 minutes at RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL detection antibody working solution to each well Incubate 10 minutes at RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL SAv HRP to each well Incubate 10 minutes at RT Dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Again dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL OptiGlow working solution to each well Incubate for 15 minutes at RT a Caution Observe the wells during the incubation When the substrate has completely drained from all wells remove the plate and pad from the holder Discard the pad Wipe the bottom of the plate with a Kimwipe to remove any liquid on the bottom surface of the plate Step 13a will be completed within the 15 minute substrate incubation time Place the plate in the reading
56. s with single aspiration step for rapid reagent transfers General setup for using an electronic multi channel pipette e Select pipette capable of delivery 5 uL amp 30 uL e g with volume range of 5 120 uL e Choose Reverse Pipetting in function setting e Use Multiple Dispensing mode to transfer the solution into the Optimiser microplate For example to transfer capture antibody solution in to a full Optimiser microplate set the program for 12 times dispensing 5 uL per dispensing Then the pipette will automatically aspirate 60 uL of solution and dispense 5 uL volumes 12 times Users will not need to move pipette back and forth to transfer solution Page 6 of 35 Multichannel pipette must be used for transferring solution into the Optimiser plate If the pipette tip is pushed inside the through hole the tip may cause the sealing tape at the base of the Optimiser to de laminate and lead to flow failure If the pipette tip does not touch the surface of well the solution may stick on the pipette tip end and not dispensed into the well OR may lead to air bubbles Small variations in flow rates time to empty well do not affect assay performance The incubation step smoothes out any flow variation differences An electronic multi channel pipette can allow for loading all reagents with a single aspiration step Ideally suited for processing multiple Optimiser microplates in paral
57. ser microplate Page 2 of 35 MATERIALS PROVIDED AND REQUIRED Materials Provided Optimiser Microplate Adoption Kit provides the critical materials and reagents necessary for the Tutorials described in this manual and for the user to develop and optimize an ELISA assay on the Optimiser microplate Table 1 identifies the kit contents their function and their required storage temperature It is recommended that the package be opened and various components stored separately as listed in Table 1 Table 1 Materials Provided with the Optimiser Microplate Adoption Kit Optimiser Holder 1 ptm Holds Optimiser Microplate and Optimiser Pad in proper alignment Contains microfluidic reaction chambers Storage Handling _ before and _ after opening Optimiser de A Microplate 10 Six Optimiser microplates for protocols described in Room manual Four spare Optimiser microplates temperature Optimiser Pad 20 Absorbs used reagent volume single use op WEN pOIYDTODYIEnE 3 For dilutions and reagent reservoir v bottom plate ee 1 vial each Coating buffer panel for screening to determine optimal Bind A L aL hy 4 mL coating buffer for capture antibody OptiBind H 1 vial 10 mL Coating buffer for model IL 6 assay A Blocking buffer and diluent for detection antibody and OptiBlock 1 vial 30 mL SAv HRP Optiwash 1 vial 60 mL Wash buffer Refrigerated x 3 2 8 C OptiGlo
58. th OptiBlock in a v shape reservoir Select a concentration which gives the max signal between 10 to 50 of RFU max From results for Illustrative IL 6 assay a concentration of 50 pg mL would be used for this experiment Blank zero Prepare 1 mL of OptiBlock in a v shape reservoir Detection Antibody Three concentrations of detection antibody working solution will be tested a same b 2 times and c 4 times as that used in conventional assay Prepare the detection antibody working solution by diluting the detection antibody stock in OptiBlock to make 200 uL final working solution Use a single column in the polypropylene 96 well v bottom plate load 90 uL of working solution in to each well Load wells A and B with 4x concentration detection antibody solution wells C and D with 2x concentration wells E and F with same as conventional concentration SAv HRP Use SAv HRP stock solution provided in the kit Prepare the SAv HRP working solution by adding 8 uL of SAv HRP stock solution to 1 2 mL of OptiBlock 1 150 dilution in a v shape reservoir Substrate solution The procedure requires 10 uL of the working substrate solution for each assay well to be used Prepare the substrate working solution in a v shape reagent reservoir by mixing 0 9 mL of OptiGlow A 0 9 mL of OptiGlow B and 18 uL of OptiGlow C OptiWash OptiWash is provided in ready to use form No further preparation is required
59. time will offer a stable assay response Furthermore using automation systems it is possible to control the dispense times precisely and incubation times can be further reduced to less than 5 min for Optimiser based assays This is less efficient in terms of capture efficiency but allows for tremendous time savings The entire assay can take less than 30 minutes Please contact Siloam s technical support for assistance Ultra Low Sample Volume 2 uL ELISA Protocol The typical assay protocol requires 5 uL on Optimiser microplate in each sample addition It is possible to further reduce the sample consumption down to 2uL without loss in sensitivity Please see application note in Siloam s web site or contact Siloam s technical support for assistance OTHER ASSAY FORMATS ON THE OPTIMISER MICROPLATE Indirect Immunoassay Siloam has developed protocols for indirect ELISA on Optimiser microplate Please contact Siloam s technical support for assistance Competitive Enzyme Immunoassay EIA Siloam has also developed protocols for competitive ELISA on Optimiser microplate Please contact Siloam s technical support for assistance Page 31 of 35 TROUBLESHOOTING The Optimiser microplate has been designed and manufactured to ensure problem free sample analysis However Siloam Biosciences has prepared the following guidance for trouble shooting that might be encountered Problem Possible Cause Soluti
60. w A 1 vial 5 mL OptiGlow B 1 vial 5 mL Components of chemifluorescent substrate OptiGlow C 1 vial 1 mL Red dye solution 1 vial 7 mL Dyed blocking buffer solution for pipetting exercise Green dye solution 1 vial 7 mL Dyed wash buffer solution for pipetting exercise IL 6 standard 1 vial Lyophilized recombinant IL 6 protein for model assay ROU standard curve 2 80 After IL 6 Capture a 1 Vial IL 6 Detection 1 vial Antibody 1 vial Captures IL 6 on solid phase Binds captured IL 6 biotin conjugated Binds detection antibody interacts with substrate to yield chemifluorescence signal 1 150 diluted with OptiBlock to make working solution reconstitution standard must be aliquoted and stored at lt 20 C Avoid repeated freeze thaw cycles for standard Material Safety Data Sheets MSDS are available on the Siloam Biosciences web site http www siloambio com Page 3 of 35 Materials Required for Testing but Not Supplied With Optimiser Microplate Adoption Kit 1 Eppendorf or similar tubes for centrifugation and dilutions 2 Kimwipes or other laboratory tissue paper 3 Reagent reservoirs V shape reservoir 4 Pipette tips for delivering in the ranges of 1 10 10 100 and 100 1000 uL Equipments Required 1 Pipette capable of accurately and precisely delivering liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipette capable of accurately and pr
61. y recommends use of an automated fluidic handler automated pipetting station for the ultra high sensitive protocol The data in the figure below illustrates the sensitivity and dynamic range obtained using the standard Optimiser ELISA procedure a single 5 uL sample addition and the improvement in sensitivity that is gained by performing 20 consecutive 5 uL sample applications to individual reaction chambers using an automated pipetting station Each additional sample incubation is 5 minutes in length Thus with 95 additional minutes of assay time the total assay time is approximately 3 hours with a corresponding increase in assay sensitivity of 20 fold The repeat sample loading methods is a reliable and simple method to tune the sensitivity of the assay to the desired range simply by adjusting the number of sample addition and incubation steps Contact Siloam Biosciences for additional details and specific guidance on running this alternate protocol 100000 5uLsample E5 LL sample repeatedly load 20 times 10000 RFU 1000 100 0 1 1 10 100 1000 Human IL 6 picogram mL Figure 10 Ultra high sensitive human IL 6 sandwich ELISA using repeat sample loading technique with Optimser microplate in conjunction with an automated pipetting station Page 33 of 35 A PLEASE CONTACT TECHNICAL SUPPORT FOR ASSISTANCE WITH THIS PROTOCOL The description provided here should not be used a formal protocol

OPV-IL6WDR - Siloam Biosciences

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