OMA-H-IL17A-02
Contents
1. Pp fat 2 3s 4 ft sf 6 500 pg ml Unused wells ina Std 2 Sample 2 Sample 10 3 reviously used plate can Std 3 Sample 3 Sample 11 O be used in a subsequent 125 pg mL assay Simply replace the D Ba p Sample 4 Sample 12 Shaded cells not used in this assay used absorbent pad The pg mL 31 3 pg mL prevents movement of Std 6 Sample 6 Sample 14 aa DE l 15 6 pe mL iquids between wells F G Std 7 Sample 7 Sample 15 7 8 pg mL 4 Optimiser plate to which standards samples and blank will be dispensed Calculations 1 Calculate the mean background signal from the blank wells wells containing Standard Diluent only at the sample incubation step 2 Subtract the mean background signal from the signal of individual standard sample and control wells 3 Create a standard curve by plotting the standard concentration x axis vs the background adjusted signal y axis Draw a best fit curve through the points of the graph A five parameter logistic curve fit with appropriate software is recommended 4 Interpolate the Hu IL 17A concentration of individual sample and control wells from the standard curve using the appropriate sample dilution factor as required Page 14 of 20 5 Note Sample concentrations should be derived by interpolation from within the standard curve range Dilute samples if necessary so that sample signal falls within the range of the standard curve 6 Calculate the mean concentration of ea2. Vortex gently to ensure thorough mixing of the reconstituted standard Refer to the enclosed Certificate of Analysis CofA for the concentration of the reconstituted standard Use freshly prepared material on the day of reconstitution or Prepare single use aliquots by dispensing reconstituted standard to appropriately sized polypropylene vials and store frozen at lt 20 C Use single use aliquots one time only on the day of thawing Avoid repeated freeze thaws b Working Solution The concentration of the reconstituted rHu IL 17A standard is specified in the CofA enclosed with each assay kit Prepare a 500 pg mL standard Standard 1 by diluting the rHu IL 17A standard appropriately in Standard Diluent Refer to CofA for dilution instruction Vortex the 500 pg mL standard briefly to mix c Standard Curve Prepare the remaining rHu IL 17A standards by performing six serial two fold dilutions in Standard Diluent beginning with the 500 pg mL standard as follows Dispense 200 uL of Standard 1 500 pg mL to well A1 of the 96 well polypropylene v bottom plate Dispense 100 uL Standard Diluent to each of the seven wells of the same column immediately below the 500 pg mL containing well wells B1 H1 Transfer 100 uL of the 500 pg mL standard from well A1 to well B1 immediately below it Page 9 of 20 The Certificate of Analysis includes instructions for the reconstitution of the lyophilized standard and f
3. 13 Calculati S enana a a a be Sanwa iepcbns s O eal nan A te anise 14 Typic DIa aaa a a a ee NO POM a a a PEE EN Med efor eT eS 15 EU eA A A a ate nice ec rsa E A E N E E EAN T RO S 15 Preckionrana RECOVERY onana ia N a T a A a aA a a A AT 15 EMICO CLC CUIOR arn a a A a te cathe tated ante a A a tate ce niadad ice 15 Detection or Native Protein eenias tease esac veduedhacuicca heen E 2a veda siaveuaann N veiw Maced eendauias 16 piget o Egress dia a AEA E I PE E E T AEA elles E E E EE 16 Alternative OptiMax ELISA Procedures soraa a a A N ake 18 Symbol indicates helpful tips to achieve optimal performance A Symbol indicates mandatory step required to ensure proper operation Intended Use Optimiser microplates and OptiMax ELISA kits Products 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 User Manuals 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 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 non conforming product during the warranty per
4. 15 9 84 The percent recovery ranged from 87 to 106 mean 97 Limit of Detection The Limit of Detection LOD minimum detectable dose MDD was determined by performing 20 replicates of Standard Diluent blank alone and calculating the mean signal 2 standard deviations of the 20 values The LOD is defined as the Hu IL 17A concentration corresponding to the mean assay blank 2 SD The LOD was determined to be lt 1 95 pg ml Page 15 of 20 Detection of Native Protein TH17 cell supernatant was obtained from cell cultures following an established induction protocol The cell culture supernatant was assayed for endogenous human IL 17A using the OptiMax Hu IL 17A ELISA and a validated conventional ELISA for Hu IL 17A with comparable results Specificity The capture and detection antibodies used in this ELISA kit are non competing monoclonal antibodies with reactivity to Hu IL 17A and no reactivity to Hu IL 17F This assay kit is reactive with the Hu IL 17A homodimer and Hu IL 17A F heterodimer Troubleshooting The Optimiser technology and OptiMax ELISA kits have been designed and manufactured to ensure problem free sample analysis However Siloam Biosciences has prepared the following guidance for trouble shooting problems that might be encountered due to the unique features of the Optimiser technology as well as problems that can be encountered with immunoassays in general Table 5 Trouble Shooting Guidelin
5. in alignment The microplate can be mounted on the holder in one orientation only The pad must be oriented correctly with the smooth surface tape side facing the holder and the absorbent surface touching the microplate 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 If bubbles are accidentally dispensed created they can be easily disrupted using a clean 26 gauge needle or similar clean sharp tipped object Accurate and Precise Delivery of 5 uL Volumes Optimiser assays require the accurate and precise delivery of 5 uL volumes The following guidance is offered to users 1 Use multichannel and single channel pipettors for which the upper limit of their operating range is lt 10 uL 2 Use pipet tips appropriate for 5 uL pipetting 3 To aspirate liquid hold the pipettor nearly vertical and immerse the pipet tip in the liquid to a depth of approximately 2 mm Withdraw the operating button slowly and steadily Wait 1 second Withdraw the tip from the liquid 4 To dispense liquid hold the pipettor nearly vertical With the pipet tips touching the surface of the Optimiser well depress the operating button slowly and steadily until the liquid is dispensed 5 Note The pipet tip must make contact with the well surface for proper dispensing see RIGHT frame below Do not pipe
6. plate 6 columns 96 Wells full plate 12 columns The incubation times for Optimiser based assays are 10 20 minutes in length Preparing all of the reagents samples and standards in advance will allow for proper timing especially for first time users DO NOT SUBSTITUTE OTHER BUFFERS OR REAGENTS FOR THOSE PROVIDED WITH THE KIT OptiMax buffers are specially formulated to work with the Optimiser microplate Substituting other buffers or reagents may lead to poor assay performance 3 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 detection antibody and SAv HRP 4 Standard Diluent Standard Diluent SD is used to reconstitute the lyophilized rHu IL 17A standard and for the preparation of rHu IL 17A standards 1 7 SD is also the diluent for Hu IL 17A controls and for samples where sample dilution is required SD is dispensed to the blank wells during the sample incubation step It is provided ready to use 5 Recombinant r Hu IL 17A standard a Stock Solution The rHu IL 17A standard is provided in lyophilized form Vi Vil Reconstitute the lyophilized standard by adding 420 uL of Standard Diluent Mix by gentle swirling until all of the lyophilized material has dissolved
7. Rl RP TR R R te NIN I NMI MTN NM Nh Materials Required for Testing but Not Supplied With OptiMax ELISA Kit 1 Eppendorf or similar polypropylene tubes for centrifugation and dilutions 0 22 um filters for sample filtration if required Kimwipes or other laboratory tissue paper Reagent reservoirs V shape reservoir Pipet tips for delivering in the ranges of 1 10 10 100 and 100 1000 uL ae ae Equipment Required Pipettor capable of accurately and precisely delivering 5 uL Multichannel pipettor capable of accurately and precisely delivering 5 uL Additional pipettors for delivery of liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipettor capable of delivering 30 uL Vortex mixer Microplate fluorescence reader and control software Analytical software Microcentrifuge capable of 13 000 rom OS OY a eS S Page 5 of 20 Unique Considerations for Optimiser Microplate The operation sequence for immunoassays performed using Optimiser microplates is very similar to that for immunoassays performed using conventional microplates By paying attention to a few key details listed here users can ensure quality results and high success The Optimiser Plate and Assembly _ Optimiser Plate _ Op timiser Pad Figure 2 Optimiser y Optimiser Holder microplate assembly Position absorbent pad on f holder align the Optimiser microplate and press d
8. User Manual OptiMax Human IL 17A ELISA Kit For the quantitative determination of human interleukin 17A IL 17A in cell culture supernatants Catalogue Numbers OMA H IL17A 02 OMA H IL17A 10 OMA H IL17A 50 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 User Manual in its entirety before using the OptiMax Hu IL 17A ELISA Kit Page 1 of 20 Table of Contents ITE CN CRON a ted eres ews recreates etek E T S 3 Materials Provided aucctaryychetneceotacavasahetsvic sblacuainhetante a a sana eb auavicamaenmenavasava A 4 Materials Required for Testing but Not Supplied With OptiMax ELISA Kit ccccsscccccsseccceessececeesececeeeeceeeeneccesauseeesaeneces 5 Eduipmen REGUE O cencia E acetan ease donsbeeauacesinesaeeo ose E ie cndeunteusnras ae eeseees 5 Unique Considerations Or Optimiser Microplate is sssstaitere Savers ot atoetiauaen T A 6 The Optimiser Plate and ASSemDy i rerent rnei aA NE NE E N A E A ANER 6 Pipetune TOF Optimiser ASSAYS urn A a A N 6 FRACS Mr S eNe aa aaa a a a acid ahah a thcaneeenteeeseeaade 7 Principleof Method eraai SHO ser orl a a oe NS seed a aa a 8 REACENE PRED a FaAtlON cicsescanssenbasce tess a aN a a sane vamakos a a Casa ioonksaduanlca N 8 POG CCU waza cheeastrnaancie anesthe ate tamaeaanaiaeaaate lt naats aro tatancinn hactettoleuai ectqnhataesencis hacen caaahe enue
9. ace within the unique Optimiser plate architecture Briefly monoclonal anti Hu IL 17A capture antibody is immobilized on the internal surfaces of the plate s microchannels Following a flush step which is equivalent to a wash step in conventional plates any unreacted sites on the microchannel surface are blocked with a blocking solution Recombinant r Hu IL 17A standard control and samples are diluted in Standard Diluent and dispensed to the Optimiser wells Hu IL 17A present in standards controls and samples will be specifically captured on the microchannel surface by the immobilized capture antibody Following another flush a biotin labeled monoclonal anti Hu IL 17A detection antibody is added to the wells The biotin labeled antibody will bind Hu IL 17A that has been captured and immobilized on the microchannel surface thus sandwiching the Hu IL 17A between the capture and detection antibodies Following another flush horseradish peroxidase labeled streptavidin SAv HRP is added to the Optimiser wells The SAv of SAv HRP binds specifically to the biotin moiety of the biotin labeled antibody if it is present in the capture antibody Hu IL 17A detection antibody complexes formed and immobilized on the microchannel surface Following two additional flushes a chemifluorescent substrate is added to the wells If horseradish peroxidase has been captured on the microchannel surface during the sequence of reactions cited above t
10. ash time substrate incubation time and read time accounted for a typical assay can be completed within approximately 2 hours Figure 1 Optimiser microplate The Optimiser microplate is a revolutionary new microplate format With an ANSI SBS compliant 96 well layout the Optimiser integrates the Power of Microfluidics to allow for low volume rapid and sensitive immunoassay protocols Figure 1 shows the Optimiser microplate schematic with magnified view of one cell of the Optimiser Each cell of the Optimiser 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 allows the well to be drained but each liquid is trapped in the microchannel by capillary forces As the next liquid volume is added 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 Page 3 of 20 Materials Provided OptiMax Hu IL 17A ELISA kits provide the critical materials and reagents necessary for the measurement of Hu IL 17A in tissue culture supernates Tables 1 and 2 Table 1 identifies the kit contents their function and their required storage temperature Table 2 restates the kit contents and indicates their indi
11. ch sample Typical Data Siloam Biosciences has validated the OptiMax Hu IL 17A ELISA kit Data acquisition and analysis utilized GenS software Excel and Graphpad Prism A summary of the validation results follows Standard Curve The rHu IL 17A standard curve ranges from 7 8 to 500 pg mL Concentration x axis and signal y axis are plotted on Log scales A typical standard curve is presented below pg ml RFU Subtracted 250 a355 a27 i as 201 2373 1 10 100 1000 human IL 17AA pg mL Figure 8 rHu IL 17A Standard Curve with Tabulated Data Precision and Recovery Validation samples were prepared by spiking rHu IL 17A into RPMI medium supplemented with 10 fetal bovine serum Each sample was tested in 24 replicates in each of four independently performed assays Both Intra and inter assay precision were determined by calculating the mean concentration standard deviation SD and percent coefficient of variation CV for each of the samples The recovery of the OptiMax Hu IL 17A ELISA assay was determined by comparing the concentration determined using the OptiMax ELISA kit with the known Hu IL 17A concentration of the validation samples as follows Percent Recovery determined concentration actual concentration x 100 Table 4 Intra assay and Inter assay Precision of OptiMax Hu IL 17A ELISA Intra assay precision Inter assay precision Standard deviation 17 6 14 5 66 38 2
12. d corresponding products Table 3 Required Filters for BioTek FLx800 Fluorescence Reader The use of an automatic multi channel pipette simplifies operation and minimizes potential for bubbles If the pipet 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 pipet tip does not touch the surface of well the solution may stick on the pipette tip and not be dispensed into the well OR may lead to air bubbles Small flow rate variations minor variations in time required for liquid to drain from wells do not affect assay performance The incubation step smoothes out any flow variation differences The Optimiser has an ANSI SBS compliant BioTek Part Number Wavelength 7082247 528 20 nm or similar layout Z axis adjustment is not required for reading the Optimiser plate Use the same setting used for a conventional 96 well microplate 7082224 590 35 nm or similar Page 7 of 20 For the FLx800 instrument and the filters listed above a sensitivity setting of 45 is recommended for the reader For more detailed information and technical support for BioTek instruments or Gen5 software please contact BioTek Instruments at 1 888 451 5171 Principle of Method The OptiMax Hu IL 17A ELISA procedure is a chemifluorescent immunoassay in which traditional ELISA reactions take pl
13. d 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 must be completed within the 15 minute substrate incubation time Page 13 of 20 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 In rare cases lt 1 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 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 14 Place the plate in the reading chamber of a fluorescence microplate reader Promptly at the conclusion of the 15 minute incubation read the plate Figure 7 Illustrative example for plate assay te SERES ee a ae a a a a a a a 1 9 a a a a a a a 2 10 a a a a a a a 3 11 Std 4 Samp Samp a a ee a a a a a a a a a 5 13 D a a fF 6 14 op eT 7 15 D eer 8 16 lt 5 uL of standard sample and blank are transferred from individual wells of polypropylene v bottom plate to duplicate cells of Optimiser
14. e in columns illustrated below o o a ee ee ee ee ee A sdi Sampled B Std2 Sample2 C Std3 Sample3 D Std4 Sampe4 E std5 Sampe5 J F std6 Samples G std7 Sampe7 J H Blank Samples 7 Detection Antibody The procedure requires 5 uL of the working detection antibody solution for each assay well to be used a Prepare a 1 250 dilution of the detection antibody stock in OptiBlock in a clean polypropylene tube according to the directions in the following table Vol of Number of Detection Volume of Wells Antibody OptiBlock Stock 48 Wells Dispense 55 uL of the working solution into 1 2 plate A each well of a single column in the Final Preparation 6 columns polypropylene 96 well v bottom plate Dispense 120 uL of the working solution into 96 Wells each well of a single column in the full plate polypropylene 96 well v bottom plate or 12 columns transfer the entire volume of working solution into a v shape reagent reservoir 8 SAv HRP The procedure requires 5 uL of the working SAv HRP solution for each assay well to be used a The SAv HRP provided with the kit is a stock solution The stock SAv HRP must be diluted with OptiBlock on the day of use to create a working The Certificate of Analysis solution includes instructions for the preparation of the SAv HRP working solution b Calculate the amount of SAv HRP working solution required
15. es Possible Cause e Disrupt the bubble with a clean 26 gauge needle A bubble is in the well e Follow recommended pipetting guidelines e Prepare excess reagent to avoid aspirating air e Do not use detergents Sample contains particulates e Centrifuge sample for 10 min at 13 000 RPM or Liquid does not drain from the p p l e Filter the sample using a 0 2 um filter Optimiser well or does not e Ensure that the absorbent side rough of the drain within 10 minutes pad is in contact with Optimiser and the tape side smooth is facing down to touch holder e Ensure the topside of the pad is touching the bottom of Optimiser plate by pushing down firmly on the 4 corners of the plate e Ensure the plate and pad are securely aligned in the holder e Use standard on the day of its reconstitution or Standard has degraded e Thaw single use aliquots fresh on each test day e Avoid repeated freeze thaws e Confirm filters meet requirements for Incorrect reader filters substrate sto a e Use within specified expiration period Antibodies or SAv HRP are p p P e Store according to recommended storage degraded temperature Substrate was prepared e Thaw OptiGlow C thoroughly before incorrectly preparing substrate working solution Substrate working solution e Prepare substrate no more than 30 minutes has degraded before plate is read Plate has lost contact with the absorbent pad or is positioned incorrectly No
16. for the assay to be performed 5 uL per well sufficient excess Page 10 of 20 c Prior to beginning the assay dilute the SAv HRP stock solution with OptiBlock according to the directions in the CofA to create the appropriate volume of SAv HRP working solution 9 Substrate solution The procedure requires 10 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 1 parts respectively according to the following table and vortex gently to mix Number Volume Volume Volume a pemarstiGn of Wells OptiGlow A OptiGlow B OptiGlow C p Dispense 100 uL of the 48 Wells working solution into each 1 2 plate 0 45 mL 0 45 mL 9 uL well of a single column in the polypropylene 96 well v bottom plate Dispense 200 uL of the working solution into each well of a single column in 96 Wells the polypropylene 96 well full plate oa Samy la v bottom plate or transfer the entire volume of working solution into a v shape reagent reservoir 10 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 a Dispense OptiWash buffer to a v shaped reagent reser
17. he enzyme will react with the substrate solution and will yield a chemifluorescent 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 Hu IL 17A in standards controls and samples and will be quantifiable when the plate is read using a microplate fluorescence reader Reagent Preparation Bring all reagents to room temperature before use and prepare all necessary dilutions before beginning the test procedure 1 OptiBind OptiBind is provided in a ready to use form No further preparation is required Do not substitute other coating buffers for OptiBind 2 Capture Antibody The procedure requires 5 uL of capture antibody working solution for each assay well to be used a Prepare the capture antibody working solution by diluting the capture antibody stock 1 31 25 in OptiBind in a clean polypropylene tube according to the following table Vol of Capture Antibody OptiBind Stock Dispense 55 uL of the working solution into each well 16 uL 0 5 mL of a single column in the polypropylene 96 well v bottom plate Dispense 120 uL of the working solution into each well of a single column in the polypropylene 96 well 32 uL 1 mL v bottom plate or transfer the entire volume of working solution into a v shape reagent reservoir Page 8 of 20 Number of Wells to be Used Final Preparation 48 Wells 1 2
18. iod is limited to replacement of or refund for the non conforming product Page 2 of 20 Introduction Interleukin 17 IL 17 is a pro inflammatory cytokine produced by a subset of T helper cells that develops distinct from the Th1 and Th2 cell differentiation pathways IL 17 also known as CTLA 8 stimulates induction of other pro inflammatory cytokines TNF alpha IL 1 beta IL 6 and IL 8 TGFB differentiation and IL 23 expansion are required for induction and maintenance of Th17 IL 17 producing cells which in turn induce the other proinflammatory cytokines IL 17 32kDa protein is produced and exists as a homo dimer has homology to a herpes virus early protein is one of the six members IL 17A F of this cytokine family and is well characterized and highly expressed by activated effector memory T cells Siloam Biosciences Human Hu IL 17A OptiMax ELISA Kit offers a rapid and sensitive chemifluorescent based ELISA procedure for Hu IL 17A that requires exceedingly small sample volumes The speed sensitivity and small sample requirements are enabled by the unique microfluidic design of the Optimiser plate The standard immunoassay reactions such as analyte capture and detection occur within a 5 uL microfluidic reaction chamber The unique microchannel geometry and small reaction volume favor rapid reaction kinetics The Hu IL 17A procedure utilizes a 5 uL sample and each reaction step is completed in 10 20 minutes With w
19. iser 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 All assay reagents for the OptiMax are provided by YSIMGENEX under agreement QuantaRed substrate is supplied by Thermo Fisher Scientific Inc SILOAM Siloam Biosciences Inc 413 Northland Blvd Cincinnati OH 45240 Phone 513 429 2976 DOC ID OPTI 2 MS 0036 A1 Fax 513 429 2946 Wwww siloambio com Page 20 of 20
20. or preparation of Standard 1 To ensure accurate preparation of the standard pipet at least 10 ul of the stock standard using an appropriate pipettor The standard curve preparation described here is an illustrative example using the first column of a v bottom plate For subsequent use the 2 or additional columns of the v bottom plate may be used Sample reagent prep in the v bottom plate is highly recommended to allow easy transfer of materials to the Optimiser using a multi channel pipettor iv Change tips Mix the contents of well B1 by gently aspirating and dispensing the liquid 8 10 times while avoiding the creation of significant bubbles in the well v Transfer 100 uL from well B1 to well C1 change tips and gently mix vi Continue serial dilutions while changing tips after each 100 uL transfer and before mixing until the 7 8 pg mL standard has been created in the seventh well well G1 of the column vii Do not transfer rHu IL 17A to the eighth well H1 It contains Standard Diluent only and will provide material for the blank wells 6 Samples Prepare samples for testing by diluting samples if required in Standard Diluent a Sample concentrations should be derived by interpolation from within the standard curve range Dilute samples if necessary so that sample signal falls within the range of the standard curve b Dispense 60 uL of each diluted sample into a single well of the v bottom plat
21. own gently to click lock the plate in holder Pipetting for Optimiser Based Assays Avoiding Bubbles While Pipetting 1 Bubbles will compromise the performance of Optimiser based assays by interfering with the flow of liquid within the microchannels 2 In particular the Standard Diluent and OptiBlock reagents may form bubbles readily if incorrectly pipetted 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 pipettor to the second stop refer to illustration below b Immerse the pipet tip in the liquid to be pipetted to a depth of about 2 mm and slowly and steadily release the operating button completely c Withdraw the tip from the liquid touching it against the edge of the reservoir to remove excess liquid d Dispense the liquid into the Optimiser loading well by gently and steadily pressing the pipettor s operating button to the first stop Briefly hold the button in this position e With the button in this position move the tip from the receiving well to the source of the liquid to be pipetted immerse the tip in the liquid and aspirate Pipetting Step Figure 3 Ready Position 1 2 3 4 First Stop t Second Stop Reverse Pipetting Procedure Page 6 of 20 Both the microplate and holder have standard markings A H rows 1 12 columns to aid
22. proceed to the next step N7 Again dispense 30 uL OptiWash to the wells Wait 10 minutes to proceed to the next step NY Dispense 10 uL OptiGlow working solution to the wells Incubate 15 minutes at RT Ny Determine the fluorescence of the wells using a microplate reader Page 12 of 20 Procedure 1 10 TL 12 13 Assemble the Optimiser Plate Optimiser Pad and Optimiser Plate Holder a Place the Optimiser Plate Holder on the laboratory bench with the Optimiser logo facing the user b Note that the top and bottom surfaces of the absorbent pad differ from one another The top side has an absorbent surface whereas a thin plastic film covers the bottom side of the pad c Place the Optimiser Pad on the Optimiser Plate Holder with the bottom side of the pad facing down on the Optimiser Holder surface d With the absorbent side of the Figure 6 Proper Alignment of the pad facing up place the Qptimiser Holder Optimiser Optimiser plate on top of the Pad and Optimiser Plate pad e Carefully align the plate holder pad and plate and push the plate down firmly using thumbs and index fingers on the 4 plate corners until the plate snaps in place on the holder 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 plate within one minute at each
23. programming Eur J Immunol In press PMID 19499530 3 Manel N Unutmaz D and Littman D R 2008 Human Th17 cell differentiation requires TGFB and induction of the nuclear receptor RORyT Nat Immunol 9 641 9 PMID 18454151 Page 17 of 20 APPENDIX 1 Alternative OptiMax ELISA Procedures A 90 Minute OptiMax ELISA The standard OptiMax ELISA procedure as described on page 12 of this User Manual requires approximately 2 hours 125 minutes to complete Most incubation steps are 10 minutes in length with the exceptions of sample incubation 20 minutes and substrate incubation 15 minutes Siloam Biosciences has developed an alternative method that can be completed in 90 minutes The sample incubation time 20 minutes final two washes 10 minutes and substrate incubation time 15 minutes are unchanged However the remaining incubation times can be reduced from 10 minutes to 5 minutes The plot in Figure 9 illustrates the adsorption kinetics of the Optimiser showing that in 5 minutes 92 of peak adsorption or binding is completed More importantly from 5 30 min next time point the adsorption only changes from 92 to 96 In doing so the total assay time is reduced from 125 minutes to 90 minutes with no change in the performance of the method Siloam strongly recommends that only users proficient in the use of the Optimiser microplate system attempt the rapid test protocol It is especially impo
24. rates the sensitivity and dynamic range obtained using the standard OptiMax 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 a robotic sample processor 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 approximately 20 fold The repeat sample loading method is a reliable and simple method to tune the sensitivity of the assay to the desired range simply by adjusting the number of sample additions and incubation steps 10000 1000 5 uL sample E5 uL sample repeatedly load 20 times 100 T l L i 0 1 1 10 100 1000 human IL17AA pg mL Figure 10 Ultra sensitive assay using repeat sample loading technique with the OptiMax Human IL 17A ELISA kit with a robotic sample processor Contact Siloam Biosciences for additional details Page 19 of 20 PLEASE CONTACT TECHNICAL SUPPORT FOR ASSISTANCE WITH THIS PROTOCOL The description provided here should not be used in place of a formal protocol Additional technical assistance is available under the Technical Support tab on the Siloam Biosciences web site http siloambio com e Material Safety Data Sheets MSDS e Using Optimiser Immunoassay Microplate Video e Optim
25. rature Refrigerated 2 8 C The reconstituted standard must be aliquoted and frozen on the day of reconstitution It is recommended that the package be opened and various components stored separately as listed in Table 1 to conserve refrigerator shelf space All materials to be refrigerated are contained in a smaller box within the product package Table 2 Materials Provided with the OptiMax ELISA Kit Quantity per assay kit per Product No Number of Units Kit Type Product Product No Product No Product No Number OMA H IL17A 02 OMA H IL17A 10 OMA H IL17A 50 2 plate kit 10 plate kit 50 plate kit Material Provided Volume Unit Optimiser Holder OPH 10 or Optimiser Pad OPH 50 96 well v bottom plate OPT FL 231 Standard Diluent 20mb vial OM 059 DP 2 Y 0 _ __ e e NIN OptiBind G_ 10mL vial oM os2g 1t J 2 O10 OptiBlock 30ml vial _OmM 055_ 1t J 2 O10 OptiWash 60ml vial _OM 054 1t J 2 10 __ OptiGlow A 5mb vial _OM 056 1t J 2 10 __ OptiGlow B 5mb vial _OM 057 t J 2 O10 OptiGlow C 1mb vial _OM 058 1t J 2 O0 Capture antibody 40ul vial OmM601102 ot J 2 10 Detection antibody 25ul vial OM601202 1t 2 10 thu IL 17A standard Lyophilized Om601302_ 2 J 10 50 Material Safety Data Sheets MSDS are available on the Siloam Biosciences web site http www siloambio com NIP
26. rtant to ensure that pipetting for each step is completed within 30 seconds It is also critically important to maintain consistency in pipetting and incubation intervals when using the accelerated protocol Contact Siloam Biosciences for additional details and specific guidance on running this alternate protocol Direct Coating of FITC Labeled Protein In Optimizer Microfluidic Chamber dle gt 90 in 5 minutes gt 75 in 10 seconds Normalized Adsorption on O NO 0 10 20 30 40 50 60 70 Residence Time in Minutes Y axis normalized to 60 minute signal Figure 9 Adsorption characteristics of capture antibody on the Optimiser microchannel surface Page 18 of 20 PLEASE CONTACT TECHNICAL SUPPORT FOR ASSISTANCE WITH THIS PROTOCOL The description provided here should not be used in place of a formal protocol APPENDIX 1 Continued An Ultrasensitive OptiMax ELISA Procedure 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 ultralow sensitivity is required The additional sample applications can be performed manually for a limited number of repeat sample loads but Siloam strongly recommends the use of a robotic sample processor for the ultra high sensitive protocol The data in the figure below illust
27. signal or unexpectedly low signal Page 16 of 20 Incorrect reader filters with overlapped wavelength bandwidth e Confirm filters meet requirements for substrate Unexpectedly high signal l ana p VAGADI e Avoid cross contamination in reagents Always Reagent contamination change the pipet tips when handling different buffers reagents Pipetting error technique or e Follow recommendations for pipetting small Poor precision l equipment volumes l l l e Follow guidelines for in plate serial two fold Curve is nonlinear Pipetting ion e Use standard on the day of its reconstitution or Degraded standard e Thaw single use aliquots fresh on each test day e Avoid repeated freeze thaws e Use within specified expiration period Degraded capture antibody e Store according to recommended storage temperature Signal of lower standard s are lt 0 following background subtraction Technical Assistance If you require assistance please contact Siloam Biosciences Inc Technical Support at 513 429 2976 or techsupport siloambio com References 1 Sundrud M S Koralov S Feuerer M Calado D P Kozhaya A E Rhule Smith A Lefebvre R E Unutmaz D Mazitschek R Waldner H Whitman M Keller T and Rao A 2009 Halofuginone inhibits Th17 cell differentiation by activating the amino acid starvation response Science In press PMID 19498172 2 Unutmaz D 2009 RORC2 the master of human Th17 cell
28. step To accomplish this first place the materials to be transferred in the enclosed 96 well polypropylene v bottom plate Then transfer the materials to the Optimiser wells using a multi channel pipettor capable of accurate and precise delivery of 5 and 10 uL volumes See Figure 7 Dispense 5 uL capture antibody working solution to the required number of wells in the Optimiser plate 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 rHu IL 17A standards controls samples 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 10 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 draine
29. t directly into the hole at the bottom of the well see WRONG frame immediately below Figure 4 Pipet tip positioning for dispensing in the Optimiser 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 i e minor variations in the time required for all liquid to drain from wells do not affect assay results Reader Settings OptiMax ELISA procedures are compatible with standard fluorescence plate readers and multi mode microplate readers with fluorescence reading capability The Technical Support section on Siloam s website offers detailed guidance on set up of the BioTek FLx800 instrument and general guidance for other readers Siloam Biosciences has verified the compatibility of OptiMax ELISA assays using OptiGlow chemifluorescence substrate in combination with BioTek Instruments FLx800 Fluorescence Microplate Reader Siloam Biosciences uses the following wavelengths an
30. vidual product numbers and the amount of each component provided per kit Refer to the enclosed Certificate of Analysis CofA for expiration dating Table 1 Materials Provided with the OptiMax ELISA Kit Storage Handling Refrigerated 2 8 C unopened rHu IL 17A standard Optimiser Holder Optimiser Plate Contains microfluidic reaction chambers Absorbs used reagent volume Optimiser Pad 96 well v bottom plate Standard Diluent Diluent for lyophilized standard standard curve and samples OptiBind OptiBlock OptiWash Washing solution G OptiGlow A OptiGlow B OptiGlow C Capture antibody Captures Hu IL 17A on solid phase Detection antibody Binds captured Hu IL 17A SAv HRP Name Function and Storage Condition Unopened Kit Contains all provided materials Store at 2 8 C Storage of Opened and Reconstituted Materials Construction of rHu IL 17A standard curve Holds Optimiser Plate and Optimiser Pad in proper alignment For dilutions and reagent reservoir Diluent for capture antibody Blocking solution and diluent for detection antibody and SAv HRP Components of chemifluorescent substrate Binds detection antibody interacts with substrate yielding chemifluorescence Page 4 of 20 Reconstitute per directions in CofA After reconstitution aliquot and store at lt 20 C Avoid repeated freeze thaws Room tempe
31. voir according to the following table Number of Volume of Final Preparation Wells OptiWash i 48 Transfer 5 mL of OptiWash into a v shaped reagent reservoir 1 2 plate 5 mL 6 columns 96 full plate 12 columns Transfer 10 mL of OptiWash into a v shaped reagent reservoir Page 11 of 20 OptiGlow C must be thoroughly thawed to function effectively Warm the reagent in a 37 C incubator or water bath or by rotating the vial gently between one s hands Figure 5 Schematic Procedure Assemble the Optimiser plate and the Optimiser pad in the Optimiser plate holder NY Dispense 5 uL of capture antibody to the required number of wells in the Optimiser plate Incubate 10 minutes at room temperature RT NY Dispense 5 uL of OptiWash to the wells Wait 10 minutes to proceed to the next step Ny Dispense 5 uL of OptiBlock to the wells Incubate 10 minutes at RT Ny Dispense 5 uL of standard control sample and blank to the wells Incubate 20 minutes at RT N7 Dispense 5 uL of OptiWash to the wells Wait 10 minutes to proceed to the next step NY Dispense 5 uL of detection antibody to the wells Incubate 10 minutes at RT Ny Dispense 5 uL of OptiWash to the wells Wait 10 minutes to proceed to the next step Dispense 5 uL of SAv HRP to the wells Incubate 10 minutes at RT Ny Dispense 30 uL of OptiWash to the wells Wait 10 minutes to
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