R-PLEX Antibody Sets Singleplex Assays

R-PLEX Antibody Sets Singleplex Assays 17379-v1-2007Mar

MSD R-PLEX Platform R-PLEX Antibody Sets Singleplex Assays Use for the development of R-PLEX singleplex assays. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. MESO SCALE DISCOVERYP A division of Meso Scale Diagnostics, LLC. 1601 Research Blvd. Rockville, MD 20850 USA www.mesoscale.com MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, R-PLEX, S-PLEX, T-PLEX, U-PLEX, V-PLEX, STREPTAVIDIN GOLD, MESO, www.mesoscale.com, SMALL SPOT (design), 96 WELL 1, 4, 7, 9, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), R-PLEX (design), S-PLEX (design), T-PLEX (design), U-PLEX (design), V-PLEX (design), It s All About U, and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC. 2017 Meso Scale Diagnostics, LLC. All rights reserved. 18183-v3-2017Dec 2

Table of Contents Introduction... 4 Principle of the Assay... 5 Components... 6 Reagents Provided... 6 Plates and Reagents for Separate Purchase... 6 Additional Materials and Equipment... 8 Safety... 8 Best Practices... 9 Reagent Preparation... 10 Assay Protocol... 12 Assay Performance... 12 Appendix A: Assay/Antibody Diluent Combinations... 13 Summary Protocol for R-PLEX Singleplex Assays... 14 Plate Diagrams... 15 Contact Information MSD Customer Service Phone: 1-240-314-2795 Fax: 1-301-990-2776 Email: CustomerService@mesoscale.com MSD Scientific Support Phone: 1-240-314-2798 Fax: 1-240-632-2219 Attn: Scientific Support Email: ScientificSupport@mesoscale.com 18183-v3-2017Dec 3

Introduction R-PLEX Antibody Sets provide an expanded menu of electrochemiluminescence assays for biomarker discovery and development. The sets include a matched biotinylated capture and SULFO-TAG conjugated detection antibody pair and a calibrator for the quick and easy development of highly sensitive immunoassays on MSD plates. R-PLEX Antibody Sets enable the development of singleplex and multiplex immunoassays: Singleplex assays on MSD GOLD Small Spot Streptavidin plates. Custom multiplex assays with combinations of R-PLEX analytes, as well as R-PLEX plus U-PLEX analytes combined. Multiplex assays use a 10-spot U-PLEX MULTI-SPOT plate and unique Linkers. This product insert provides the details of developing a singleplex assay on the MSD platform using R-PLEX Antibody Sets. A complete list of available R-PLEX Antibody Sets is available at www.mesoscale.com/r-plex. A representative data set for each R-PLEX Antibody Set is presented in the product-specific datasheets available at www.mesoscale.com/r-plex-documents. 18183-v3-2017Dec 4

Principle of the Assay Singleplex assays can be easily developed on MSD GOLD Small Spot Streptavidin plates. These plates provide high sensitivity, consistent performance, and excellent inter- and intra-lot uniformity. The R-PLEX Antibody Set includes a biotinylated capture antibody that binds to streptavidin on the plate surface. Analyte in the sample binds to the capture reagent; a detection antibody conjugated with an electrochemiluminescent label (MSD GOLD SULFO-TAG label) binds to the analytes to complete the sandwich immunoassay (Figure 1). Once the sandwich immunoassay is complete, the plate is loaded into an MSD instrument where a voltage applied to the plate electrodes causes the captured labels to emit light. The instrument measures the intensity of emitted light (which is proportional to the amount of analyte present in the sample) and provides a quantitative measure of the analyte in the sample. Figure 1. R-PLEX singleplex assay on a MSD GOLD Small Spot Streptavidin Plate 18183-v3-2017Dec 5

Components The following tables list the components needed for assay development with R-PLEX Antibody Sets. Reagents Provided The R-PLEX Antibody Set contains a biotinylated capture antibody, a SULFO-TAG conjugated detection antibody, and a frozen calibrator. The calibrator is provided at a 20-fold higher concentration than the suggested top-of-the-curve concentration. The topof-the-curve concentration for each assay is shown in the product-specific datasheet. Table 1. Contents of R-PLEX Antibody Set Name Storage Size Quantity Supplied Description Biotin Capture Antibody 5 Plates 1 vial Biotinylated capture antibody. Provided as one vial 2 8 C (analyte-specific) 50 Plates 10 vials per five plates. SULFO-TAG Detection Antibody 5 Plates 1 vial SULFO-TAG conjugated detection antibody (100X). 2 8 C (analyte-specific) 50 Plates 10 vials Provided as one vial per five plates. Calibrator 5 Plates 5 vials Native or recombinant protein, provided frozen in a -70 C (analyte-specific) 50 Plates 50 vials buffered diluent. Provided as one vial per plate. Plates and Reagents for Separate Purchase MSD offers a range of plates and reagents to enable assay development using R-PLEX Antibody Sets. Plates and reagents are also available for individual purchase, and in different pack sizes. For a complete listing of all available assay development plates and reagents, visit our website at www.mesoscale.com. Plates Table 2. MSD GOLD singleplex plates Name 1 Plate 5 Plates 30 Plates 120 Plates 510 Plates MSD GOLD 96-well Small Spot Streptavidin Plates L45SA-1 L45SA-2 L45SA-5 L45SA-6 L45SA-7 Note: Singleplex assays can be developed on MSD streptavidin- or avidin-coated plates. For more information, refer to MSD GOLD Streptavidin and Avidin Plates product insert available at www.mesoscale.com. MSD GOLD Read Buffer Table 3. MSD GOLD Read Buffer Name Storage Catalog # Description MSD GOLD Read Buffer, 200 ml 15 30 C R92TG-2 MSD GOLD Read Buffer, 1000 ml 15-30 C R92TG-1 Buffer to catalyze the electrochemiluminescence reaction. Provided at the working concentration of the assay. Sufficient for 10 plates. Buffer to catalyze the electrochemiluminescence reaction. Provided at the working concentration of the assay. Sufficient for 50 plates. 18183-v3-2017Dec 6

Diluents R-PLEX assays may have specific diluents for sample and calibrator dilution as well as for preparation of the detection antibody solution. Refer to the R-PLEX product-specific datasheet supplied with the product for the diluents tested in the assay. The datasheet is also available at www.mesoscale.com/r-plex-documents. The catalog numbers for commonly used diluents in singleplex assays are provided in Table 4, but a range of diluents are available for purchase at www.mesoscale.com. See Appendix A for more information on the diluents and diluent combinations tested in R-PLEX singleplex assays. Table 4. Common diluents used in R-PLEX singleplex assays Name Catalog # Size Diluent 1 R50CK-4 R50CK-2 1 R51BB-4 8 ml Diluent 2 R51BB-3 40 ml R51BB-2 200 ml Diluent 3 R50AP-1 8 ml R50AP-2 40 ml Diluent 7 R54BB-4 5 ml R54BB-3 Diluent 8 R54BA-4 5 ml R54BA-3 Diluent 10 R55BB-5 10 ml R55BB-3 Diluent 11 R55BA-4 5 ml R55BA-3 Diluent 12 R50JA-4 10 ml R50JA-3 Diluent 13 R56BB-4 10 ml R56BB-3 Diluent 27 R50OA-3 30 ml R50OA-2 1 Name Catalog # Size Diluent 35 Diluent 37 Diluent 40 Diluent 41 Diluent 42 Diluent 43 Diluent 45 Diluent 100* Diluent 101 R50AE-3 R50AE-2 R50AF-3 R50AF-6 R50AJ-1 R50AJ-2 R50AH-1 R50AH-2 R50AK-1 R50AK-2 R50AG-1 R50AG-2 R50AI-3 R50AI-4 R50AA-4 R50AA-2 R50AA-3 R51AD-3 R51AD-7 30 ml 1 25 ml 125 ml 5 ml 40 ml 10 ml 10 ml 10 ml 8 ml 40 ml 200 ml 1,000 ml 700 ml *Diluent 100 can be used as a coating diluent instead of 0.5% BSA in PBS. Note: To run five plates, of assay diluent and 40 ml of antibody diluent are required when assaying samples that are diluted up to 10-fold (40 samples per plate, run in duplicate). Additional assay diluent is necessary for samples that are diluted greater than 10-fold. Diluent 100 may be used in place of assay diluent for samples that require high dilution. Testing of different diluents can help optimize assays for specific experimental conditions. Wash Buffer Table 5. Catalog number of MSD Wash Buffer Name Storage Catalog # Size Description MSD Wash Buffer (20X) Room temperature R61AA-1 100 ml Phosphate-buffered saline (PBS) plus 0.05% Tween-20 (PBS-T) for plate washing Note: This size of MSD Wash Buffer is sufficient for washing 4 plates manually or for washing 2 plates with an automated plate washer. Prepare a 1X working solution. For 1 plate, combine 15 ml of MSD Wash Buffer (20X) with 285 ml of deionized water. 18183-v3-2017Dec 7

Additional Materials and Equipment Appropriately sized tubes for reagent preparation Polypropylene microcentrifuge tubes for preparing dilutions Liquid handling equipment suitable for dispensing 10 to 150 µl/well into a 96-well microtiter plate Plate washing equipment: automated plate washer or multichannel pipette Microtiter plate shaker (rotary) capable of shaking at 500 1,000 rpm Adhesive plate seals Deionized water Vortex mixer Safety Use safe laboratory practices: wear gloves, safety glasses, and lab coats when handling assay components. Handle and dispose of all hazardous samples properly in accordance with local, state, and federal guidelines. Additional product-specific safety information is available in the safety data sheet (SDS), which can be obtained from MSD Customer Service or at www.mesoscale.com. 18183-v3-2017Dec 8

Best Practices Bring frozen diluent to room temperature in a 22-25 C water bath. Thaw frozen calibrator (when applicable) on wet ice. Prepare Calibrator Standards and samples in polypropylene microcentrifuge tubes. Use a fresh pipette tip for each dilution and mix by vortexing after each dilution. Avoid prolonged exposure of the detection antibody (stock or diluted) to light. During the antibody incubation step, plates do not need to be shielded from light (except for direct sunlight). Avoid bubbles in wells during all pipetting steps as they may lead to variable results. Bubbles introduced when adding MSD GOLD Read Buffer may interfere with signal detection. Use reverse pipetting when necessary to avoid the introduction of bubbles. For empty wells, pipette gently to the bottom corner. Plate shaking should be vigorous, with a rotary motion between 500 and 1,000 rpm. Binding reactions may reach equilibrium sooner if you use shaking at the middle of this range (~700 rpm) or above. When using an automated plate washer, rotate the plate 180 degrees between wash steps to improve assay precision. Gently tap the plate on a paper towel to remove residual fluid after washing. If an incubation step needs to be extended, leave the sample or detection antibody solution in the plate to keep the plate from drying out. Remove the plate seal prior to reading the plate. Make sure that the MSD GOLD Read Buffer is at room temperature when added to a plate. Do not shake the plate after adding MSD GOLD Read Buffer. To improve inter-plate precision, keep time intervals consistent between adding MSD GOLD Read Buffer and reading the plate. Unless otherwise directed, read the plate as soon as possible after adding MSD GOLD Read Buffer. If the sample results are above the top of the calibration curve, dilute the samples and repeat the assay. When running a partial plate, seal the unused sectors to avoid contaminating unused wells. Remove all seals before reading. Partially used plates may be stored up to 30 days at 2 8 C in the original foil pouch with desiccant. You may adjust volumes proportionally when preparing reagents. Calibrators for R-PLEX Antibody Sets whose representative curves surpass 1 million counts may be diluted an extra 4-fold to lower the top signals. 18183-v3-2017Dec 9

Reagent Preparation Bring all reagents to room temperature and refer to the Best Practices section before beginning the protocol. Important: Upon first thaw, aliquot diluents into suitable volumes before refreezing. To prepare supplemental reagents such as MSD Wash Buffer, please refer to the Components section. Coat Plate Add 200 µl of biotinylated capture antibody to 3.3 ml of coating diluent. Mix by vortexing. Note: Coating diluents can be simple diluents containing 0.5% BSA in PBS or use MSD Diluent 100. Add 25 µl of the above solution to each well of the MSD GOLD Small Spot Streptavidin plate. Tap the plate gently on all sides. Seal the plate with an adhesive plate seal and incubate at room temperature for 1 hour or at 2-8 C overnight. Shaking the plate during incubation is required. Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T (PBS plus 0.05% Tween-20). The plate is now coated with capture antibody. Prepare Calibrator Standards MSD supplies calibrator for R-PLEX analytes at a concentration that is 20-fold higher than the recommended highest standard. We recommend a 7-point calibration curve with 4-fold serial dilution steps and a zero calibrator blank. Thaw the stock calibrator and keep on ice, then add to assay diluent at room temperature to make the calibration curve solutions. To prepare 7 calibrator solutions plus a zero calibrator for up to 4 replicates: Prepare the highest calibrator by adding 15 µl of stock calibrator to 285 µl of assay diluent. Mix well. Prepare the next calibrator by transferring 50 µl of the highest calibrator to 150 µl of assay diluent. Mix well. Repeat 4-fold serial dilution 5 times to generate 7 calibrators. Use assay diluent as the blank. Discard any unused, diluted calibrators. Note: For the top-of-the-curve concentration of Calibrator, refer to the product-specific datasheet supplied with the R-PLEX Antibody Set. The datasheet is also available at www.mesoscale.com/r-plex-documents. Figure 2. Dilution schema for Calibrator Standards for R-PLEX singleplex assays 18183-v3-2017Dec 10

Prepare Detection Antibody Solution The detection antibody is provided as a 100X stock solution. The working solution is 1X. Prepare the detection antibody solution immediately prior to use. For one plate, combine: 60 µl of the supplied 100X detection antibody 5,940 µl of antibody diluent Dilute Samples Depending on the sample set under investigation, a dilution may be necessary. Recommendations on dilution factor for different sample types are provided in the R-PLEX product-specific datasheet included in the product box. Assay diluent may be used for sample dilution. The dilution factor for a given sample type needs to be optimized. Note: Additional assay diluent is necessary for samples that are diluted greater than 10-fold. Diluent 100 may be used in place of assay diluent for samples that require high dilution. Prepare MSD GOLD Read Buffer MSD provides MSD GOLD Read Buffer at the working concentration of the assay. Equilibrate the read buffer to room temperature before use. To avoid bubbles, do not vortex. Note: If using MSD Read Buffer T (4X), dilute the buffer two-fold in deionized water to make a 2X working solution. 18183-v3-2017Dec 11

Assay Protocol Note: Before beginning STEP 1, prepare the plate as described on p. 10. STEP 1: Add Samples and Calibrators Add 25 µl of assay diluent to each well. Tap the plate gently on all sides. Add 25 µl of the prepared Calibrator Standard or sample to each well. Seal the plate with an adhesive plate seal. Incubate at room temperature with shaking for 1 hour. STEP 2: Wash and Add Detection Antibody Solution Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 50 µl of detection antibody solution to each well. Seal the plate with an adhesive plate seal. Incubate at room temperature with shaking for 1 hour. STEP 3: Wash and Read Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 150 µl of MSD GOLD Read Buffer (or 2X MSD Read Buffer T) to each well. Analyze the plate on an MSD instrument. Incubation in read buffer is not required before reading the plate. Alternate Protocols The suggestions below may be useful for simplifying the protocol. Alternate Protocol 1, Extended Incubation: Incubating samples overnight at 2 8 C may improve sensitivity for some assays. Alternate Protocol 2, Reduced Wash: For cell culture supernatants, you may simplify the protocol by eliminating one of the wash steps. After incubating the Calibrator Standard or sample, add detection antibody solution to the plate without decanting or washing the plate. Assay Performance A representative data set for each assay is presented in the product-specific datasheet shipped with the product; also available at www.mesoscale.com/r-plex-documents. The data represent performance of the assay tested in singleplex format on U-PLEX plates. The data were generated during the development of the assay and do not represent the product specifications. Under your experimental conditions, the assay may perform differently than the representative data shown. 18183-v3-2017Dec 12

Appendix A: Assay/Antibody Diluent Combinations Below is additional information on the diluent combinations that have been tested with R-PLEX Antibody Sets. Please refer to the R-PLEX datasheet for the diluent combination used for your analyte(s) of interest. These diluent combinations are recommended as starting points when developing your assay; however, other diluent combinations should be considered based on the sample environment. For further guidance, please contact our Scientific Support team at scientificsupport@mesoscale.com. Human Assay Diluent 2 Antibody Diluent 3 Alpha-amylase 1 100 CA125 20 CA15-3 20 CA50 20 CD5* CD27 CTLA-4 2 E-Cadherin 20 Galectin-3 50 Granzyme A 2 Granzyme B 2 LAG3 2 MET (soluble) 20 MIG 4 β-ngf 2 Osteonectin 20 Osteoprotegerin 2 PD1 2 PD-L2 2 Resistin 16 TLR1 2 TNF-RII 10 *Please review datasheet. Assay Diluent 7 Antibody Diluent 3 Calbindin 10 MMP-1 10 MMP-3 10 MMP-9 10 Osteoactivin 10 P-Cadherin 10 TNF-RI 10 Assay Diluent 7 Antibody Diluent 7 Enolase 2 5 PRDX6 5 Assay Diluent 7 Antibody Diluent 8 Fas (soluble) 50 IL-6R 50 Mesothelin 50 Osteocalcin 50 RANTES 50 Assay Diluent 7 Antibody Diluent 11 FGF (basic) 2 PlGF 2 Tie-2 2 VEGF-D 2 Assay Diluent 7 Antibody Diluent 37 Annexin A1 4 CEA 4 Osteopontin 4 PRDX-1 4 PSGL-1 4 TFF3 4 Assay Diluent 7 Antibody Diluent 101 BDNF 5 Assay Diluent 10 Antibody Diluent 3 Ang-1 2 Ang-2 2 Cytokeratin-8 2 E-Selectin 2 FasL 2 ICAM-3 2 Nectin-4 2 P-Selectin 2 SCF 2 Thrombomodulin 2 Assay Diluent 12 Antibody Diluent 11 PYY (active)* 2 *Please review datasheet. Assay Diluent 12 Antibody Diluent 12 A2M 4,000 B2M 4,000 BAFF-R 2 CD31/PECAM-1 20 Corin/ATC2 2 Cystatin C 100 GITR 2 GITRL 2 GLP-1 (active) 4 GLP-1 (inactive) 4 GLP-1 (total) 4 MMP-7 2 OX40 2 OX40L 2 Pentraxin 3 2 S100A8/MRP8 2 S100A12 2 Serpin A1 4,000 Serpin A12/Vaspin 2 Tenascin C 100 TNFRSF10C 20 vwf 1,000 Assay Diluent 13 Antibody Diluent 12 Complement factor D 100 C-Peptide FGF-21 12,000 FGF-23 4 FSH GIP (active)* 2 GIP (inactive)* 2 GIP (total)* 2 Insulin Leptin LH Prolactin 10 *Please review datasheet. Assay Diluent 37 Antibody Diluent 37 AGP 300,000 ApoA1 300,000 ApoE 4,000 Complement C3 300,000 Haptoglobin 300,000 RBP4 2,000 Assay Diluent 43 Antibody Diluent 3 Adiponectin 8,000 BCMA 400 Endoglin 2 gp130 (soluble) 25 MIP-4 1,000 MPO 20 PYY (total) 4 Assay Diluent 100 Antibody Diluent 100 BAFF 8 Assay Diluent 101 Antibody Diluent 27 GFAP 2 Assay Diluent 101 Antibody Diluent 37 CA1 5,000 Calprotectin 100 Cathepsin D 100 Clusterin 5,000 Complement C9 5,000 DPPIV 5,000 FAP-α/SEPR 100 GDF-15 100 Gelsolin 100 TfR-1 (soluble) 100 TIMP-1 100 Assay Diluent 101 Antibody Diluent 100 ApoC3 4,000 Assay Diluent 101 Antibody Diluent 101 VILIP-1 2 Special Handling Required LRRK2 Please review LRRK2 (ps935) datasheet. Rat Assay Diluent 42 Antibody Diluent 40 EPO GM-CSF 2 IL-1α 4 IL-1β 2 IL-2 4 IL-6 2 IL-10 IL-13 8 KC/GRO 4 MCP-1 MIP-3α TNF-α 2 VEGF-A NHP Assay Diluent 2 Antibody Diluent 3 Alpha-amylase 2A 5,000 Note regarding sample dilutions: This table gives recommended dilutions for serum and plasma samples. Additional assay diluent is necessary for samples that are diluted greater than 10-fold. Diluent 100 may be used in place of assay diluent for samples that require high dilution. Testing of different diluents can help optimize assays for specific experimental conditions. 18183-v3-2017Dec 13

Summary Protocol for R-PLEX Singleplex Assays Gather Required Assay Components R-PLEX Antibody Set MSD GOLD Small Spot Streptavidin plate Coating diluent, assay diluent, and antibody diluent MSD GOLD Read Buffer MSD Wash Buffer or PBS-T (PBS plus 0.05% Tween-20) Coat Plate Add 200 µl of biotinylated capture antibody to 3.3 ml of coating diluent. Mix by vortexing. Add 25 µl of the above solution to each well of an MSD GOLD Small Spot Streptavidin plate. Tap the plate gently on all sides. Seal the plate with an adhesive plate seal and incubate at room temperature with shaking for 1 hour or at 2-8 C overnight Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. The plate is now coated and ready for use. Assay Protocol STEP 1: Add Samples and Calibrators Add 25 µl of assay diluent to each well. Tap the plate gently on all sides. Add 25 µl of prepared Calibrator Standard or sample to each well. Seal the plate with an adhesive plate seal. Incubate at room temperature with shaking for 1 hour. STEP 2: Wash and Add Detection Antibody Solution Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 50 µl of detection antibody solution to each well. Seal the plate with an adhesive plate seal and incubate at room temperature with shaking for 1 hour. STEP 3: Wash and Read Wash the plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 150 µl of MSD GOLD Read Buffer (or 2X MSD Read Buffer T) to each well. Analyze the plate on an MSD instrument. Incubation in read buffer is not required before reading the plate. 18183-v2-2017Nov 14

Plate Diagrams 18183-v3-2017Dec 15