USER MANUAL SUPPLEMENT:
Contents
1. ___ Following the incubation dispense 5 uL OptiWash to each well Incubate 10 minutes at RT ___ Following the wash step dispense 5 uL OptiBlock to each well Incubate 10 minutes at RT ___ Following the block step dispense 5 uL standard or sample to each well Incubate 20 minutes at RT __ Following the sample incubation dispense 5 uL OptiWash to each well Incubate 10 minutes at RT __ Following the wash step dispense 5 uL of the detection antibody working solution to each well Incubate 10 minutes at RT __ Following the detection antibody step dispense 5 uL OptiWash to each well Incubate 10 minutes at RT ___ Following the wash step dispense 5 uL of the SAv HRP working solution to each well Incubate 10 minutes at RT ___ Following the SAv HRP incubation dispense 30 uL OptiWash to each well Incubate 10 minutes at RT __ Immediately following step 10 dispense 30 uL OptiWash to each well for a second 30 uL wash Incubate 10 minutes at RT ___ Dispense 10 uL OptiGlow working solution to each well Incubate 15 minutes at RT a ___ Observe the plate periodically during this incubation When the substrate solution has drained from all wells first remove the plate from the holder and then remove the pad from the plate b ___ Wipe the bottom of the plate thoroughly with a KimWipe or similar laboratory tissue ___ Read the plate using an excitation wavelength of 528 20 nm an2. USER MANUAL SUPPLEMENT p gt i owered By Assay Solution for Invitrogen Human c Met Total Antibody Pair PTI MISER Invitrogen Catalog Number CHOO285 An ELISA for human Hu c Met Total utilizing reagents from LIFE Technologies Invitrogen Catalog Number CH0O0285 has been successfully transferred from a conventional 96 well plate format to the Optimiser microplate platform to achieve the following key performance benefits Sample Volume 5 l Assay time Total assay time 2 hours 3 5 hour savings Assay reagents 90 saving in antibody use 10 assays for the cost of 1 Sensitivity Range Equivalent sensitivity 0 78 50 ng ml e Potential to increase sensitivity to 0 04 ng ml e Potential to achieve gt 2 log dynamic range 0 78 580 ng ml Intended Use This User Manual Supplement is intended to be used in conjunction with Invitrogen s Technical Data Sheet for the Human c Met Total Antibody Pair Catalog Number CHOO0285 employed in this Optimiser based ELISA procedure Please refer to the vendor s instructions for material storage preparation and concentration information The procedure described in this User Manual Supplement is intended as a starting reference for the investigator using the vendor s assay reagents with the Optimiser microplate system Siloam has optimized this procedure using OptiBlock as blocking solution as reagent diluent and as the media to reconstitute the protei
3. Standard 8 Anti Rabbit Anti Rabbit IgG HRP sis HRP Optimiser Materials Material Product Number Optimiser plate with holder OPH 10 OptiMax buffer reagent pack with substrate OMR 10 H Other Material amp Equipment Materials Equipment Polypropylene centrifuge tubes 1 5 2 mL Fluorescence plate reader ei cap pet ps o tips orien meee mixer a C E channel Single channel pipettor s asd E EETA Test tube rack o rack Multichannel Multichannel pipetior 96 well OA v bottom 96 well polypropylene v bottom plate s Reagent reservoirs Reagent reservoirs v bottom ss bottom 1 Refer to the respective Technical Data Sheets TDS for storage concentration and other relevant information i Optimiser plates and corresponding OptiMax buffer reagents are also available in 2 plate and 50 plate configuration Page 3 of 13 3 4 3 6 7 REAGENT PREPARATION 1 2 Working concentrations of all materials should be prepared before beginning the procedure Capture antibody working solution a Refer to the vendor s TDS for the protein concentration of the stock capture antibody solution b Prepare the capture antibody working solution by diluting the stock capture antibody 1 125 in OptiBind H Lyophilized Standard a Reconstitute the lyophilized protein standard with OptiBlock OB Refer to the vendor s TDS for further directions the concentration of the r
4. d an emission wavelength of 590 35 nm ___ This space is provided as a simple way of documenting the completion of each step Page 8 of 13 Page 9 of 13 Calculations 1 Calculate the mean background signal RFU 2 Subtract the mean background signal from the individual standard and sample values 3 Calculate the mean background adjusted signal for each standard and sample 4 Prepare a standard curve by plotting the concentration of the standard on the x axis and the background adjusted signal on the y axis using a 4 parameter curve fit 5 Interpolate the sample concentrations from the standard curve Calculate the final concentration after applying the sample dilution factor if applicable Typical Data The standard curve illustrated below was generated using the method reagents and equipment specified in this procedure Optimiser Based ELISA for Human c Met Total Bkg Adjusted RFU 0 10 20 30 40 50 Human c Met Total ng mL Page 10 of 13 Page 11 of 13 Additional technical assistance is available under the Technical Support tab on the Siloam Biosciences web site http www siloambio com Material Safety Data Sheets MSDS Using Optimiser Immunoassay Microplate Video Optimiser User s Guide Reader Settings Quick Reference Guide Frequently Asked Questions Application Notes Two additional videos appear under the Technology tab of the web site Optimiser Principles of O
5. econstituted standard and storage conditions Standard Curve a Prepare standard 1 by diluting the reconstituted standard to 50 ng mL in OptiBlock b Dispense 200 uL of standard 1 to well A1 of the polypropylene 96 well v bottom plate c Dispense 100 uL of OptiBlock to each of the 7 wells immediately below well A1 d Prepare serial two fold dilutions of the standard by successive 100 uL transfers through well G1 Change pipet tips after each transfer and mix the well contents 8 10 times by gently aspirating and dispensing the well contents Do not transfer standard to well H1 Well H1 will serve as the assay blank 0 pg mL 200 uL Std 1 100 uL OB 100 uL OB 100 uL OB 100 uL OB 100 uL OB 100 uL OB 100 uL OB Detection antibody a Refer to the vendor s TDS for the protein concentration of the stock detection antibody solution b Prepare the detection antibody working solution by diluting the stock detection antibody material 1 125 in OptiBlock Horseradish Peroxidase Labeled HRP Anti Rabbit IgG a Refer to the vendor s TDS or vial label for the dilution factor required to prepare the anti rabbit lIgG HRP working solution b Prepare the anti rabbit IgG HRP working solution by diluting the stock material appropriately in OptiBlock OptiGlow working solution a Prepare the OptiGlow substrate working solution by combining OptiGlow A OptiGlow B and OptiGlow C in proportions of 50 50 1 parts re
6. es Optimiser based ELISAs use a unique flush step rather than the traditional and laborious wash step used in conventional ELISAs To flush the user simply dispenses OptiWash into the Optimiser well The wash buffer flushes the used reagent sample from the microchannel into an absorbent pad beneath the plate The Optimiser flush is equally effective as the traditional washes Page 6 of 13 9 Std 2 Samp Samp 2 10 gt ee ie jae 3 11 Std 4 Samp Samp o fee ELS ioe ise 5 13 F Std 6 Samp Samp PF LSS fora oe ee ise 7 15 Pm ye oe ae 8 16 4 Polypropylene v bottom plate containing diluted standards samples and blank 5 uL of standard sample and blank are transferred from individual wells of polypropylene v bottom plate to duplicate cells of the Optimiser plate pot at 2 3 4 5 6 7 a seen oon ae ae oer D std4 Sample 4 Sample 12 Shaded cells not used in this assay Samples Somme A A A a a fample e Sammie a a A A Sarpe w7 Sarle1s A A O a O G 0 pg mL 4 Optimiser plate to which standards samples and blank will be dispensed Page 7 of 13 PROCEDURE 1 2 10 11 12 13 ___ Assemble the Optimiser plate pad and holder as described earlier __ Dispense 5 uL of the working capture antibody solution to the appropriate number of wells in the Optimiser plate Incubate 10 minutes at room temperature RT
7. n standard Siloam has not evaluated this procedure for its applicability for analysis of tissue culture supernatants serum plasma or other sample types Use of this assay for the analysis of tissue culture supernatant serum plasma or other sample types may require further optimization of some assay parameters by the investigator for example standard curve and sample diluents to achieve desired results It is expected that investigators following this assay procedure are familiar with the Optimiser microplate system If you have not used the Optimiser microplate before please order the Evaluation kit Catalog OPV IL6 which provides a comprehensive overview to the Optimiser microplate system The Evaluation Kit guides the user through correct pipetting procedures for Optimiser microplates and contains all necessary materials and instructions for completing an illustrative human IL 6 assay Investigators are strongly urged to familiarize themselves with the Optimiser microplate system before completing the procedure described in this document Please contact Siloam s tech support techsupport siloambio com for any questions regarding this procedure FOR RESEARCH USE ONLY Not for Use in Diagnostic Procedures MATERIALS Assay Reagents Material ti oS aaa Cga Anti c Met ee Coating Antibody Anti c Met Ateher Total Detection Antibody _ Detection Antibody Invitrogen CHOO285 Per TDS c Met eMe Total sanad oO o
8. older surface and push down firmly until the plate snaps into position 2 Reverse pipetting Introduction of bubbles to the Optimiser wells will compromise method performance by occluding the microchannel To avoid introducing bubbles always use the reverse pipetting technique when delivering materials to an Optimiser well a Beginning with the pipettor s operating button in the ready position depress the operating button to the second stop See figure in step c below b Immerse the pipet tip in the liquid to be transferred Aspirate the liquid by releasing the operating button returning it to the ready position c Dispense the liquid to the Optimiser well by depressing the operating button to the first stop Ensure that the pipet tip is touching the well surface as the liquid is dispensed Pipetting Step Ready Position 1 2 3 4 First Stop l t Second Stop 3 Transferring reagents standards and samples to the Optimiser plate Due to the short incubation times it is critical that antibodies standards samples SAV HRP and substrate are transferred from their source to the Optimiser wells quickly lt 1 minute but accurately To accomplish this a First dispense the materials to a polypropylene 96 well v bottom plate b Then using a multichannel pipettor transfer the materials from the polypropylene v bottom plate to the Optimiser wells as illustrated in the figure on the next page Optimiser Wash
9. peration Running an Assay with Optimiser DOC ID ETS 1 MS 0009 A1 QuantaRed substrate is supplied by Thermo Fisher Scientific Inc Page 12 of 13 SILOAM iosciences Better Immunoassays Through Innovative Microfluidics SILOAM BIOSCIENCES INC 413 Northland Blvd Cincinnati OH 45240 USA Tel 1 513 429 2976 Fax 1 513 429 2976 http www siloambio com
10. spectively b Note Prepare the working substrate solution no more than 30 minutes before reading the plate Page 4 of 13 Use of OptiBlock This method was developed using Siloam Biosciences OptiBlock not only as blocking agent following the coating of the microfluidic reaction chamber with capture antibody and but also as diluent for the standards detection antibody and SAv HRP The intent in developing this method was to demonstrate the ease of transitioning Invitrogen s c Met Total Antibody Pair from their intended use in conventional ELISAs to an Optimiser based ELISA format Use of this method for the analysis of tissue culture supernatant serum plasma or other sample types may require further optimization of some assay parameters by the investigator to achieve desired results for example standard curve and sample diluents Siloam Biosciences has developed OptiMax Standard Diluent for use in the analysis of cell culture supernatants This product has been incorporated in Siloam Biosciences commercially available OptiMax ELISA Kits Page 5 of 13 DISPENSING MATERIALS TO THE OPTIMISER PLATE 1 Optimiser assembly Assemble the Optimiser plate pad and holder as illustrated a Position the holder on the lab bench with the Optimiser logo facing the user b The absorbent pad must be positioned with the plastic covered surface facing the holder c Position the pad and plate on the h
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