User Manual
Contents
1. Optimiser Holder NA 1 1 1 Optimiser Plate NA OPH O2 or 2 10 50 ee OPH 10 or Optimiser Pad NA OPH 50 4 20 100 96 well v bottom plate NA OPT FL 231 1 5 25 Standard Diluent 20 mL vial OM 059 1 2 10 OptiBind G 10 mL vial OM 052G 1 2 10 OptiBlock 30 mL vial OM 055 1 2 10 OptiWash 60 mL vial OM 054 1 2 10 OptiGlow A 5 mL vial OM 056 1 2 10 OptiGlow B 5 mL vial OM 057 1 2 10 OptiGlow C 1 mL vial OM 058 1 2 10 Capture antibody 40 uL vial OM606102 1 2 10 Detection antibody 25 uL vial OM606202 1 2 10 rMs IFN y standard Lyophilized OM606302 2 10 50 SAv HRP 25 uL vial OMO602 1 2 10 Material Safety Data Sheets MSDS are available on the Siloam Biosciences web site http www siloambio com Materials Required for Testing but Not Supplied With OptiMax ELISA Kit 1 Eppendorf or similar polypropylene tubes for centrifugation and dilutions oP ws Equipment Required Vortex mixer 00 SOG Re Met 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 Microplate fluorescence reader and control software Analytical software Microcentrifuge capable of 13 000 rpm Page 5 of 21 Pipettor capable of accurately and precisely delivering 5 uL Multichannel pipettor capable of accurately and precisely delivering 5 uL Additional pip2. 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 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Samp Samp 1 9 B Std 2 Samp Samp 2 10 C Std 3 Samp Samp 3 11 D Std 4 Samp Samp 4 12 E Std5 Samp Samp 5 13 F Std 6 Samp Samp 6 14 G Std 7 Samp Samp 7 15 H Blank Samp Samp 8 16 M Polypropylene v bottom plate containing diluted standards samples and blank lt 5 uL of standard sample and blank are transferred from individual wells of polypropylene v bottom plate to duplicate cells of Optimiser 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Sample 1 Sample 9 210 pg ml Unused wells in a B Std 2 Sample 2 Sample 10 previously used plate can 105 pg mL C Std 3 Sample 3 Sample 11 be used in a subsequent 53 p
3. in the holder No signal or unexpectedly low signal Standard has degraded Use standard on the day of its reconstitution or Thaw single use aliquots fresh on each test day Avoid repeated freeze thaws Incorrect reader filters 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 incorrectly Thaw OptiGlow C thoroughly before preparing substrate working solution Substrate working solution has degraded Prepare substrate no more than 30 minutes before 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 pipet tips when handling different buffers reagents Poor precision Pipetting error technique or equipment Follow recommendations for pipetting small volumes Curve is nonlinear Pipetting e Follow guidelines for in plate serial two fold dilutions Page 17 of 21 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 s
4. User Manual OptiMax Mouse IFN gamma ELISA Kit For the quantitative determination of mouse MS interferon gamma in cell culture supernatants Catalogue Numbers OMA M IFNG 02 OMA M IFNG 10 OMA M IFNG 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 Mouse IFN gamma ELISA Kit Page 1of 21 Table of Contents IPE EO CII GUI OIA Aaeeea a a a das van a aa desea fen a bcs ean sea a e a a a 3 Materials Provided soer ccs cdccisscaccectessnanectgcseelcecessnxih eb aneautevcuusonaes a aia aaisa eaaa aaiae detaupabeulesss caveuda eve des saeat tos aio iio aaia e N 4 Materials Required for Testing but Not Supplied With OptiMax ELISA Kit 0 0 cccsssssssscecsssseceecesseesesseseesasseesseseescassaeeceeaees 5 EQUIPMENT REQUINGG 2 3 ma hesse As cus teats fase a a ee ees phew haaa a dee en acdc sean s eigen sn ve a lanes 5 Unique Considerations for Optimiser Microplate oeenn neeaaea aii a ai A d E Ea a Seidi ara 6 The Optimiser Plateand Assembly torinese ieee E E E EA ENNE EEEE EENE 6 Pipetting for Optimiser Based ASSAYS cccssscccssssecssssscessessesesssessssssseeecassaecosssecsesessesesasesssssessesscaseaecasseeseesssseseasesassassaesasecesseseaseaes 6 Reader Settings nl a A a Aa a r E 7 Principle f MEt OA e e aa a e E E E E A E a aaraa ie 8 R
5. 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 NY 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 NY 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 proceed to the next step NY 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 v Determine the fluorescence of the wells using a microplate reader Page 12 of 21 Procedure 1 10 11 12 13 Assemble the Optimiser Plate Optimiser Pad and Optimiser Plate Holder a P
6. chambers Room temperature Optimiser Pad Absorbs used reagent volume 96 well v bottom aT For dilutions and reagent reservoir plate Standard Diluent Diluent for lyophilized standard standard curve and samples OptiBind G Diluent for capture antibody Blocking solution and diluent for OptiBlock detection antibody and SAv HRP OptiWash Washing solution OptiGlow A Refrigerated 2 8 C OptiGlow B Components of chemifluorescent substrate OptiGlow C Capture antibody Captures Ms IFN y on solid phase Detection antibody Binds captured Ms IFN y Binds detection antibody interacts with substrate yielding chemifluorescence SAv HRP Page 4 of 21 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 Material Provided Volume Unit Number OMA M IFNG 02 OMA M IENG 10 OMA M IFNG 50 2 plate kit 10 plate kit 50 plate kit
7. clusive remedy for non conforming product during the warranty period is limited to replacement of or refund for the non conforming product Page 2 of 21 Introduction Mouse Ms interferon gamma IFN y a cytokine is one of the most important members of the interferon IFN family It is a polypeptide of 155 amino acids having a molecular weight of 17 709 kDa Mouse IFN y is the sole type II IFN structurally unrelated to type IFNs and binds to different receptors and is encoded by a separate chromosomal locus Schroder K et al 2004 IFN y is primarily secreted by activated T cells and natural killer cells and can promote macrophage activation mediate anti viral and anti bacterial immunity enhance antigen presentation orchestrate activation of the innate immune system coordinate lymphocyte endothelium interaction regulate Th1 Th2 balance and control cellular proliferation and apoptosis Gattoni et al 2006 Cytokines most notably IL 12 and IL 18 control the production of IFN y during the course of the immune response IFN y acts mainly through a receptor mediated signaling process by IFNGR1 IFNGR2 and signals through the JAK STAT pathway Schroder K et al 2004 Siloam Biosciences OptiMax Mouse Interferon gamma ELISA Kit offers a rapid and sensitive chemifluorescent based ELISA procedure for Ms IFN y that requires exceedingly small sample volumes The speed sensitivity and small sample requirements are enabled by the unique m
8. dditional details Page 20 of 21 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 Optimiser 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 0039 A0 ee a ee www siloambio com Page 21 of 21
9. eagent Pr paration EE A E EA TE A LA A A 8 Procedure oinetan enr Re a PR a a e e a a a e aa a T a a 13 Calc latiO NSi aa a a i a a a aaia i a e a e 14 TY Pi Gal DI t PEE A EASE E EER E EE A 15 Standardi Curve am eiie aa a a a AEA ANS a a a aa a a n e a 15 Precisiom ana RECOVEIV r TA EEA aa A E AEE ob OTS 15 Limit of Detection miseniigenann a a a Ge dah a eee a nee 16 Detection of Native Protein meniran i spe spot tect a a a a a a a E oresecetetee 16 TEOUblEShOOtiIN einam i e a a a e a a ia Tae aa vee ahaa Aaaa T a Ea 17 Alternative OptiMax ELISA ProCe duress soninn E E A E AEEA A EEE e a EER Oae iea 19 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 ex
10. ettors for delivery of liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipettor capable of delivering 30 uL 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 lt _ Op timiser Pad Figure 2 Optimiser Eo Gaidee microplate assembly Position absorbent pad on holder align the Optimiser microplate and press down 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 Figure 3 Reverse Pipetting Procedure To aspirate liquid press the operating button of the pipettor to the second stop refer to illustration below Immerse the pipet tip in the liquid to be pipetted to a de
11. g 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 and corresponding products Table 3 Required Filters for BioTek FLx800 Fluorescence Reader Function BioTek Part Number Wavelength Excitation 7082247 528 20 nm or similar Emission 7082224 590 35 nm or similar Page 7 of 21 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 m
12. g mL assay Simply replace the D BA sampio 4 Sample 12 Shaded cells not used in this assay used absorbent pad The E Std 5 Sample 5 Sample 13 Optimiser design 13 pg mL prevents movement of F Std 6 Sample 6 Sample 14 EA 6 6 pe mL liquids between wells G Std 7 Sample 7 Sample 15 3 3 pg mL H Blank Sample 8 Sample 16 0 pg mL M 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 Ms IFN y concentration of individual sample and control wells from the standard curve using the appropriate sample dilution factor as required Page 14 of 21 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 each sample Typical Data Siloam Biosciences has validated the OptiMax Ms IFN y ELISA kit Data acquisition and a
13. gle channel pipettors for which the upper limit of their operating range is lt 10 uL Use pipet tips appropriate for 5 uL pipetting 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 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 Note The pipet 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 immediately below RIGHT WRONG a Ls wa Kaxa 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 The buffer reagents provided with the assay kit have been developed and validated for the Optimiser microplate Do not substitute alternate buffers or reagents 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 testin
14. he wells The biotin labeled antibody will bind Ms IFN y that has been captured and immobilized on the microchannel surface thus sandwiching the Ms IFN y 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 Ms IFN y 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 the 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 Ms IFN y 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 re
15. icrofluidic 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 Ms IFN y procedure utilizes a 5 uL sample and each reaction step is completed in 10 20 minutes With wash time substrate incubation time and read time accounted for a typical assay can be completed within approximately 2 hours This OptiMax ELISA kit has been calibrated against the R amp D Systems Quantikine Mouse IFN gamma ELISA kit Data generated using the OptiMax Mouse IFN y kit should closely correlate with that generated using the R amp D Systems Quantikine Mouse IFN gamma ELISA kit 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 trap
16. inor 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 layout Z axis adjustment is not required for reading the Optimiser plate Use the same setting used for a conventional 96 well microplate 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 Mouse IFN y ELISA procedure is a chemifluorescent immunoassay in which traditional ELISA reactions take place within the unique Optimiser plate architecture Briefly anti IFN y 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 Ms IFN y standard control and samples are diluted in Standard Diluent and dispensed to the Optimiser wells Ms IFN y 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 anti Ms IFN y detection antibody is added to t
17. lace 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 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
18. nalysis utilized GenS software Excel and Graphpad Prism A summary of the validation results follows Standard Curve The rMs IFN y standard curve ranges from 3 3 to 210 pg mL Concentration x axis and signal y axis are plotted on Log scales A typical standard curve is presented below 10000 ij 1000 100 1 10 100 1000 Mouse IFN gamma pg mL Figure 8 rMs IFN y Standard Curve with Tabulated Data mouse IFN y pg mL RFU 1 RFU 2 Mean Blank subtracted 210 7366 7720 7543 7291 105 4187 4523 4355 4103 53 2232 2492 2362 2110 26 1536 1515 1525 5 1273 13 871 892 881 5 629 6 6 516 589 552 5 300 3 3 401 421 411 159 0 221 284 252 5 Table 4 Tabulated RFU Data for Ms IFN y Standard Curve This OptiMax ELISA kit has been calibrated against the R amp D Systems Quantikine Mouse IFN gamma ELISA kit Data generated using the OptiMax Mouse IFN y kit should closely correlate with that generated using the R amp D Systems Quantikine Mouse IFN gamma ELISA kit Precision and Recovery Validation samples were prepared by spiking rMs IFN y 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 Page 15 of 21 precision were determined by calculating the mean concentration standard deviation SD and percent coefficient of variation CV for each
19. ndard Diluent Refer to CofA for dilution instruction Vortex the 210 pg mL standard briefly to mix c Standard Curve Prepare the remaining rMs IFN y standards by performing six serial two fold dilutions in Standard Diluent beginning with the 210 pg mL standard as follows Dispense 200 uL of Standard 1 210 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 210 pg mL containing well wells B1 H1 1 1 A 200 uL Std 1 A 210 B 100 uL SD B 105 C 100 uL SD C 53 D 100pLSD gt D 26 E 100 uL SD E 13 F 100 uL SD F 6 6 G 100 uL SD G 3 3 H 100 uL SD H 0 Transfer 100 uL of the 210 pg mL standard from well A1 to well B1 immediately below it Page 9 of 21 The Certificate of Analysis includes instructions for the reconstitution of the lyophilized standard and for 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
20. 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 Final Preparation of Wells OptiGlow A OptiGlow B OptiGlow C 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 oori 0 9 mL 18 pL the polypropylene 96 well full plate 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 reservoir according to the following table Number of Volume of F Wells OptiWash Final Preparation 48 1 2 plate 5 mL Transfer 5 mL of OptiWash into a v shaped reagent e columns reservoir 96 full plate 10 mL Transfer 10 mL of OptiWash into a v shaped reagent a9 columns reservoir Page 11 of 21 OptiGlow C must be thoroughly thawed to function effectively
21. of the samples The recovery of the OptiMax Moude IFN y ELISA assay was determined by comparing the concentration determined using the OptiMax ELISA kit with the known Ms IFN y concentration of the validation samples as follows Percent Recovery determined concentration actual concentration x 100 Table 5 Intra assay and Inter assay Precision of OptiMax Mouse IFN y ELISA Intra assay precision Inter assay precision Sample pg mL 158 78 8 39 4 158 78 8 39 4 Mean of calculated concentration pg ml 153 77 4 36 6 156 0 75 8 35 2 Standard deviation 11 30 6 80 3 41 6 2 5 0 1 2 CV 7 4 8 8 9 3 4 0 6 7 3 3 The percent recovery ranged from 92 to 97 mean 95 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 Ms IFN y concentration corresponding to the mean assay blank 2 SD The LOD was determined to be lt 0 8 pg ml Detection of Native Protein Six million mouse splenocytes were cultured in 3 mL of RPMI 1640 medium with the following supplements 10 fetal bovine serum 2 mM L glutamine 100 ug ml streptomycin and 100 U ml penicillin The splenocytes were stimulated with 10 ug ml of Concanavalin A incubated at 37 C 5 CO for 2 days The cell culture supernatant
22. orption 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 important 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 80 OO gt 90 in 5 minutes gt 75 in 10 seconds Normalized Adsorption gt a O O N 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 19 of 21 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 uniq
23. ped 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 21 Materials Provided OptiMax Ms IFN y ELISA kits provide the critical materials and reagents necessary for the measurement of Ms IFN y 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 individual 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 Name Function and Storage Condition Material Function Storage Unopened Kit Contains all provided materials Store at 2 8 C Storage of Opened and Reconstituted Materials Material Function Storage Handling Refrigerated 2 8 C unopened Reconstitute per directions in CofA After reconstitution aliquot and store at lt 20 C Avoid repeated freeze thaws ile Neystandare Construction of rMs IFN y standard curve Holds Optimiser Plate and Optimiser Optimiser Holder f Pad in proper alignment Optimiser Plate Contains microfluidic reaction
24. pth of about 2 mm and slowly and steadily release the operating button completely Withdraw the tip from the liquid touching it against the edge of the reservoir to remove excess liquid 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 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 Ready Position 1 2 3 4 First Stop t Second Stop Page 6 of 21 Both the microplate and holder have standard markings A H rows 1 12 columns to aid 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 sin
25. quires 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 62 5 in OptiBind in a clean polypropylene tube according to the following table Number of volt Capture Vol of F Wells to be 3 nee Final Preparation Antibody OptiBind Used Stock 48 Wells Dispense 55 uL of the working solution into each well 1 2 plate 8 uL 0 5 mL of a single column in the polypropylene 96 well v 6 columns bottom plate Dispense 120 uL of the working solution into each 96 Wells f well of a single column in the polypropylene 96 well full plate 16 uL 1mL i 43 columns v bottom plate or transfer the entire volume of working solution into a v shape reagent reservoir Page 8 of 21 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 5 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 inc
26. torage 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 Interferon gamma an overview of signals mechanisms and functions Schroder K Hertzog PJ Ravasi T Hume DA J Leukoc Biol 2004 Feb 75 2 163 89 Epub 2003 Oct 2 Review PMID 14525967 2 Interferon gamma biologic functions and HCV therapy type I II 1 of 2 parts Gattoni A Parlato A Vangieri B Bresciani M Derna R Clin Ter 2006 Jul Aug 157 4 377 86 Review Retraction in Clin Ter 2008 May Jun 159 3 207 PMID 17051976 Page 18 of 21 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 ads
27. tube according to the directions in the following table Vol of Number of Detection Volume of Final Preparation Wells Antibody OptiBlock Stock 48 Wells Dispense 55 uL of the working solution into 1 2 plate 4 uL 0 5 mL each well of a single column in the 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 8 uL 1mL 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 solution b Calculate the amount of SAv HRP working solution required for the assay to be performed 5 uL per well sufficient excess Page 10 of 21 The Certificate of Analysis includes instructions for the preparation of the SAv HRP working solution 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
28. 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 rMs IFN y 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 pL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 10 pL 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 must be completed within the 15 minute substrate incubation time Page 13 of 21 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
29. ubation with the capture antibody solution OptiBlock is also used as the diluent for the detection antibody and SAv HRP Standard Diluent Standard Diluent SD is used to reconstitute the lyophilized rMs IFN y standard and for the preparation of rMs IFN y standards 1 7 SD is also the diluent for Ms IFN y 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 Recombinant r Ms IFN y standard a Stock Solution The rMs IFN y standard is provided in lyophilized form vi vii Reconstitute the lyophilized standard by adding 420 uL of Standard Diluent Mix by gentle swirling until all of the lyophilized material has dissolved 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 rMs IFN y standard is specified in the CofA enclosed with each assay kit Prepare a 210 pg mL standard Standard 1 by diluting the rMs IFN y standard appropriately in Sta
30. ue 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 illustrates 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 Spulsample 10 mouse IFN gamma pg mL Figure 10 Ultra sensitive assay using repeat sample loading technique with the OptiMax Mouse IFN y ELISA kit with a robotic sample processor Contact Siloam Biosciences for a
31. using a multi channel pipettor 6 7 iv vi vii 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 Transfer 100 uL from well B1 to well C1 change tips and gently mix Continue serial dilutions while changing tips after each 100 uL transfer and before mixing until the 3 3 pg mL standard has been created in the seventh well well G1 of the column Do not transfer rMs IFN y to the eighth well H1 It contains Standard Diluent only and will provide material for the blank wells 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 plate in columns illustrated below 1 2 side 12 Std 1 Sample 1 Std 2 Sample 2 Std 3 Sample 3 Std 4 Sample 4 Std 5 Sample 5 Std 6 Sample 6 Std 7 Sample 7 ZA TNMs olol Blank Sample 8 Detection Antibody The procedure requires 5 uL of the working detection antibody solution for each assay well to be used a Prepare a 1 125 dilution of the detection antibody stock in OptiBlock in a clean polypropylene
32. was assayed for endogenous mouse IFN y using the OptiMax Mouse IFN y ELISA and the R amp D Systems Quantikine Mouse IFN gamma ELISA with comparable results Page 16 of 21 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 6 Trouble Shooting Guidelines Problem Possible Cause Solution 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 and the tape side smooth is facing down to touch holder Ensure the topside of the pad is touching the bottom of Optimiser plate by pushing down firmly on the 4 corners of the plate Ensure the plate and pad are securely aligned
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