U-PLEX Development Packs

Transcription

1 U-PLEX Development Packs For the development of multiplex assays 1-Plate 5-Plate 25-Plate 2-Assay K15227N-1 K15227N-2 K15227N-4 3-Assay K15228N-1 K15228N-2 K15228N-4 4-Assay K15229N-1 K15229N-2 K15229N-4 5-Assay K15230N-1 K15230N-2 K15230N-4 6-Assay K15231N-1 K15231N-2 K15231N-4 7-Assay K15232N-1 K15232N-2 K15232N-4 8-Assay K15233N-1 K15233N-2 K15233N-4 9-Assay K15234N-1 K15234N-2 K15234N-4 10-Assay K15235N-1 K15235N-2 K15235N-4 FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES v1-2007Mar

2 MSD U-PLEX Platform U-PLEX Development Pack For the development of multiplex assays using R-PLEX Antibody Sets, U-PLEX Antibody Sets, and/or your own antibodies. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. MESO SCALE DISCOVERYP A division of Meso Scale Diagnostics, LLC Research Blvd. Rockville, MD USA 1www.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, 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. 2015, 2017, 2018 Meso Scale Diagnostics, LLC. All rights reserved v5-2018Apr 2

3 Table of Contents Introduction... 4 Assay Formats... 5 U-PLEX Advantage... 6 Components... 7 Additional Components Additional Materials and Equipment Safety Best Practices Typical Protocols Assay Protocol Appendix A: Assay Optimization Appendix B: Assay Performance Appendix C: Assay/Antibody Diluent Combinations Summary Protocol for Multiplex Assays Spot Map Plate Diagrams Contact Information MSD Customer Service Phone: Fax: CustomerService@mesoscale.com MSD Scientific Support Phone: Fax: attn: Scientific Support ScientificSupport@mesoscale.com v5-2018Apr 3

4 Introduction The U-PLEX platform allows you to create your own multiplex assays. Using two simple tools, a 10-spot U-PLEX plate and unique Linkers, you can build custom multiplex panels with compatible combinations of R-PLEX and U-PLEX analytes and your own analytes. Assays can be run in a number of different formats. The most common format is the sandwich immunoassay and MSD has developed an expanding menu of R-PLEX and U-PLEX Antibody Sets that can be used to create custom multiplex assays on U-PLEX plates. This product insert provides the details for developing multiplex combinations using R-PLEX and U-PLEX Antibody Sets or using your own reagents. Principle of the Assay Biotinylated capture reagents (e.g., antibodies, peptides, proteins, nucleic acids) are coated on U-PLEX plates using U-PLEX Linkers. For sandwich immunoassays (Figure 1), biotinylated capture antibodies are coupled to U-PLEX Linkers. These Linkers self-assemble onto unique spots on the U-PLEX plate. s in the sample bind to the capture reagents; detection antibodies conjugated with electrochemiluminescent labels (MSD GOLD SULFO-TAG) bind to the analytes to complete the immunoassay sandwich. Once the sandwich immunoassay is complete, the U-PLEX 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, to provide a quantitative measure of each analyte in the sample. Figure 1. Sandwich Immunoassay on a 10-Spot plate depicting the electrochemiluminescence reaction v5-2018Apr 4

5 Assay Formats The U-PLEX platform enables you to build assays in several different formats as shown below. The most common format is the sandwich immunoassay in which a biotinylated capture antibody, specific to the analyte, is coupled to a U-PLEX Linker and coated on a U-PLEX plate (Figure 2A). Once the analyte is captured, the immunoassay can be completed using a SULFO-TAG conjugated detection antibody or a specific detection antibody followed by a SULFO-TAG conjugated secondary anti-species antibody (Figure 2B). Guidelines on antibody selection are provided in Appendix A. For your convenience, MSD has developed a large, expanding suite of R-PLEX and U-PLEX Antibody Sets that can be used in combination with U-PLEX plates to create custom multiplex immunoassays (see page 15 for detailed protocols). Immunoassays can also be performed as bridging assays, as is the case for immunogenicity assays, and can be done in either stepwise or homogenous formats depending on the assay reagents. Additionally, the U-PLEX platform can be used to measure antibodies against proteins, antibodies, peptides, carbohydrates, or polysaccharides (Figure 2C). The antigen should be biotinylated and then coupled to the U-PLEX Linker. The sample containing the antibody can be added either simultaneously with the U-PLEX-coupled Linker or after the antigen is coated on the plate. In this assay format, a SULFO-TAG anti-species antibody is used for detection. Oligonucleotides and nucleic acids can be measured with the U-PLEX platform using a direct binding format (Figure 2D). The capture oligonucleotide should be biotinylated and coupled to a U-PLEX Linker and the detection oligonucleotide should be coupled to SULFO-TAG label. Figure 2. (A) Biotinylated capture antibody used with SULFO-TAG conjugated detection antibody. (B) Biotinylated capture antibody used with unlabeled primary detection antibody and SULFO-TAG conjugated anti-species antibody as a secondary reporter. (C) Auto-antibody or serology assay format to detect the presence of antibodies against proteins, peptides, or carbohydrates. (D) Biotinylated oligonucleotides or nucleic acids used with SULFO-TAG conjugated oligonucleotides v5-2018Apr 5

6 U-PLEX Advantage The U-PLEX platform has been developed for custom multiplexing MSD assays. You can conveniently prepare a personalized plate at the bench with ease and without delay. U-PLEX assays follow a workflow similar to that of ELISAs and bead-based assays; however, the MSD platform offers many advantages over the alternative immunoassays, as shown in the table below. Note that U-PLEX assays offer a significant conservation of precious samples and antibodies with gains in sensitivity, large dynamic range, and specificity. Table 1. Comparison of U-PLEX, ELISA, and bead-based immunoassays Feature U-PLEX ELISA Bead-based Sample volume 25 µl µl 50 µl Dynamic range 3 4 logs 1 2 logs 2 3 logs Multiplex enabled Yes No Yes Simple Protocol Yes No No Number of washes (between incubation steps) Matrix effects No Yes Yes (bead aggregation) Read time 1 min 5 min 60 min Instrument maintenance None Monthly calibration Clogging problems This product insert is designed to help you develop personalized multiplex assays using MSD U-PLEX technology. Components-This section provides the listing and descriptions of components that are included in U-PLEX Development Packs. It also includes a list of additional components needed for creating R-PLEX and U-PLEX custom multiplexes. These additional components can be purchased separately. Typical Protocol-This section provides a detailed description of plate coating and an optimized immunoassay protocol using MSD R-PLEX and U-PLEX Antibody Sets (page 15) or using your own reagents (page 19). Appendix A-This section provides guidelines on various assay formats, assay protocol, and antibody optimization, as well as additional considerations for multiplexing. Appendix B-This section provides typical assay performance data obtained from multiple assays. Appendix C-This section provides information on the assay/antibody diluent combinations that have been tested with R-PLEX Antibody Sets. At the end of the document, MSD provides summary protocols and graphics that can be removed and used at the bench for your convenience. a. Summary for Plate Preparation and a typical Assay Protocol b. Spot Map for Linker assignment c. Plate Diagram for assay layout Please read the entire insert prior to performing the assay v5-2018Apr 6

7 Components 10-spot 96-well U-PLEX Plates U-PLEX plates contain 10 individual spots on the bottom of each well; the spots correspond to 10 unique U-PLEX Linkers. The number and layout of active spots on the plate depends on the number of assays to be multiplexed (Figure 3). U-PLEX Development Packs are available in 2 to 10 assay formats and come with corresponding plates (Table 2). Figure 3. Spot Map of the different U-PLEX multiplex plates showing the placement of Linkers within a well. The colored spots represent the active U-PLEX binding spots. The numbering convention for the different spots is maintained in the software visualization tools, on the plate packaging, and in the data files. You can choose the appropriate Development Pack according to the number of assays you plan to multiplex. For example, if you plan to multiplex 4 assays together, you can choose the U-PLEX Development Pack 4-Assay. This Pack includes a SECTOR plate with 4 activated spots at locations 1, 3, 8, and 10 as shown in Figure 3. Table 2. U-PLEX multiplex plates supplied with each pack size SECTOR Plates Catalog # Storage Size Description U-PLEX 2-Assay N05227A-1 U-PLEX 3-Assay N05228A-1 U-PLEX 4-Assay N05229A-1 U-PLEX 5-Assay U-PLEX 6-Assay U-PLEX 7-Assay N05230A-1 N05231A-1 N05232A C 1-Plate, 5-Plate, and 25-Plate Packs 96-well plate, foil sealed, with desiccant. U-PLEX 8-Assay N05233A-1 U-PLEX 9-Assay N05234A-1 U-PLEX 10-Assay N05235A v5-2018Apr 7

8 Linkers Based upon the Development Pack you select, you will receive 2-10 unique Linkers that correspond to the number of assays you plan to multiplex (Table 3). Each Linker has a biotin-binding domain that couples to the biotin located on the capture reagent as well as a domain that binds to its matching spot on the U-PLEX plate. The Linkers are color coded and numbered with the spot to which they attach on the plate. 1-Plate packs include 300 µl of each Linker; 5-Plate packs include 1.8 ml of each Linker. 25-Plate packs include 5 vials of 1.8 ml of each Linker. Figure 4. Unique color coded Linkers, Antibody Sets, and Calibrator vials Table 3. Linkers supplied with each U-PLEX Development Pack Name Linker 1 Linker 2 Linker 3 Linker 4 Linker 5 Linker 6 Linker 7 Linker 8 Linker 9 Linker 10 U-PLEX 2-Assay U-PLEX 3-Assay U-PLEX 4-Assay U-PLEX 5-Assay U-PLEX 6-Assay U-PLEX 7-Assay U-PLEX 8-Assay U-PLEX 9-Assay U-PLEX 10-Assay We recommend recording which antibody is coupled to each Linker when performing the coupling step (as described in the Reagent Preparation section) v5-2018Apr 8

9 Table 4. Linker color-coding, storage conditions, catalog numbers, and size Name Color-coding Storage Catalog # Size Linker 1 Linker 2 Linker 3 Linker 4 Linker 5 Linker 6 Linker 7 Linker 8 Linker 9 Linker C 2 8 C 2 8 C 2 8 C 2 8 C 2 8 C 2 8 C 2 8 C 2 8 C 2 8 C E ml 1 vial Quantity Supplied 1 Plate 5 Plates 25 Plates E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials E ml 1 vial E ml 1 vial 5 vials Other Reagents Provided with Development Pack Table 5. Additional reagents provided in the Development Pack Reagent Storage Catalog # Size Quantity Supplied 1-Plate Pack 5-Plate Pack 25-Plate Pack Stop Solution 2 8 C R50AO-1 40 ml 1 bottle 1 bottle 5 bottles Read Buffer T (4X)* RT R92TC-3 50 ml 1 bottle 1 bottle 5 bottles *Remove bottle from box and allow read buffer to equilibrate to room temperature (RT) before use. Description Biotin-containing buffer to stop Linker-antibody coupling reaction. Buffer to catalyze the electrochemiluminescence reaction. Note: MSD GOLD Read Buffer can be substituted for Read Buffer T. MSD GOLD Read Buffer is provided at the working concentration of the assay v5-2018Apr 9

10 Additional Components The following is a list of additional components needed for creating R-PLEX and U-PLEX custom multiplexes in combination with U-PLEX Development Packs. These may have been purchased separately. R-PLEX Reagents R-PLEX Antibody Sets 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 is also shown in the datasheet. Table 6. Contents of R-PLEX Antibody Set Name Storage Size Quantity Supplied Description Biotin Capture Antibody (analyte-specific) SULFO-TAG Detection Antibody (analyte-specific) Calibrator (analyte-specific) 2 8 C 2 8 C -70 C 5 Plates 1 vial Biotinylated capture antibody. Provided as one vial 50 Plates 10 vials per five plates. 5 Plates 1 vial SULFO-TAG conjugated detection antibody (100X). 50 Plates 10 vials Provided as one vial per five plates. 1 Plate 5 vials Native or recombinant protein, provided frozen in a 50 Plates 50 vials buffered diluent. Provided as one vial per plate. U-PLEX Reagents U-PLEX Antibody Sets The U-PLEX Antibody Set contains a biotinylated capture antibody and a SULFO-TAG conjugated detection antibody. Table 7. Contents of U-PLEX Antibody Set Name Storage Size Quantity Supplied Description U-PLEX Biotin Capture Antibody Biotinylated capture antibody. Provided as one vial 2 8 C 5 Plates 1 vial (analyte-specific) per five plates. U-PLEX SULFO-TAG Detection SULFO-TAG conjugated detection antibody (100X). 2 8 C 5 Plates 1 vial Antibody (analyte-specific) Provided as one vial per five plates. Note: U-PLEX Antibody Sets do not include Calibrator. The calibrators are available as multi-analyte blends and can be purchased separately. U-PLEX Calibrators For multiplexing in combination with U-PLEX assays, one or more of the following Calibrators are required, depending on the U-PLEX assays of interest. Calibrators for U-PLEX assays are supplied as lyophilized, multi-analyte blends. Individual analyte concentrations are provided in the lot-specific Calibrator certificates of analysis (COA) received with your order and available at v5-2018Apr 10

11 Table 8. s included in the Calibrator blends available for U-PLEX Biomarker Group 1 Assays Species Name Storage Catalog # Size Human Mouse Quantity Supplied 1 Plate 5 Plates 25 Plates Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials Calibrator C C vial 1 vial 5 vials 25 vials s GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, TNF-α, VEGF-A Eotaxin, Eotaxin-3, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC G-CSF, IFN-α2a, IL-1α, IL-7, IL-12/IL- 23p40, IL-15, IL-16, IL-18, TNF-β, TPO CTACK, ENA-78, Fractalkine, I-TAC, MIP-3α, MIP-3β, SDF-1α IL-17A/F, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-29/IFN-λ1, IL-31, IL-33, TSLP EPO, FLT3L, IFN-β, IL-1RA, IL-2Rα, IL-3, IL-9, IL-17B, IL-17C, IL-17D Eotaxin-2, GRO-α, I-309, MCP-2, MCP-3, M-CSF, MIF, MIP-5, TRAIL, YKL-40 EPO, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, KC/GRO, TNF-α, VEGF-A IL-16, IL-17A, IL-17C, IL-17E/IL-25, IL-21, IL-22, IL-23 Calibrator C C vial 1 vial 5 vials 25 vials IL-15, IL-17F, IL-31, IL-33, IL-7p28/IL-30 Calibrator C C vial 1 vial 5 vials 25 vials IL-9, IL-17A/F, IP-10, MCP-1, MIP-1α, MIP-1β, MIP-2, MIP-3α v5-2018Apr 11

12 General Reagents Diluents R-PLEX and U-PLEX assays may have specific diluents for sample and calibrator dilution as well as for preparation of the detection antibody solution. The catalog numbers for diluents commonly used in multiplex assays are provided in Table 9, but a range of diluents are available for purchase at Information on diluent combinations that have been tested with R-PLEX Antibody Sets is found in Appendix C. Table 9. Catalog numbers of common diluents used in R-PLEX and U-PLEX multiplex assays Name Catalog # Size Diluent 1 Diluent 2 Diluent 3 Diluent 5 Diluent 6 Diluent 7 Diluent 8 Diluent 10 Diluent 11 Diluent 12 Diluent 13 Diluent 17 Diluent 22 R50CK-4 R50CK-2 R51BB-4 R51BB-3 R51BB-2 R50AP-1 R50AP-2 R52BA-5 R52BA-6 R53BB-4 R53BB-3 R54BB-4 R54BB-3 R54BA-4 R54BA-3 R55BB-5 R55BB-3 R55BA-4 R55BA-3 R50JA-4 R50JA-3 R56BB-4 R56BB-3 R50KA-4 R50KA-3 R50BB-8 R50BB-4 50 ml 150 ml 8 ml 40 ml 200 ml 8 ml 40 ml 25 ml 125 ml 8 ml 40 ml 5 ml 50 ml 5 ml 50 ml 10 ml 50 ml 5 ml 50 ml 10 ml 50 ml 10 ml 50 ml 6 ml 30 ml 8 ml 40 ml Name Catalog # Size Diluent 27 Diluent 29 R50OA-3 R50OA-2 R50HA-4 R50HA-3 30 ml 150 ml 15 ml 40 ml Diluent 30 R50AB-4 25 ml 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 150 ml 25 ml 125 ml 5 ml 40 ml 10 ml 50 ml 10 ml 50 ml 10 ml 50 ml 8 ml 40 ml 50 ml 200 ml 1,000 ml 50 ml 700 ml Note: To run five plates, 50 ml 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. For further guidance on choosing diluents, please contact our Scientific Support team at or at scientificsupport@mesoscale.com v5-2018Apr 12

13 Wash Buffer Table 10. Catalog number of MSD Wash Buffer Name Storage Catalog # Size Description MSD Wash Buffer (20X) RT R61AA 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. MSD GOLD Read Buffer Table 11. MSD GOLD Read Buffer Name Storage Catalog # Description MSD GOLD Read Buffer, 200 ml C R92TG-2 MSD GOLD Read Buffer, 1000 ml 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. Note: MSD GOLD Read Buffer can be used in place of MSD Read Buffer T. No preparation is required prior to use. 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 and wear gloves, safety glasses, and lab coats when handling kit 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 v5-2018Apr 13

14 Best Practices Bring frozen diluent to room temperature in a C water bath. Thaw frozen calibrator (when applicable) on wet ice. For multiplexes assays, avoid cross-contamination between Linkers by following the techniques below: o Open one U-PLEX Linker vial at a time. Close the cap after use. o Each Linker vial is color coded; ensure that each cap and tube have matching colors when opening and closing vials. o Use filtered pipette tips. o Use a fresh pipette tip after each reagent addition. For long-term studies using multiple plates of the same assay, it is recommended that the same Linker be coupled with the same antibody for the duration of the study. 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 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 you plan to coat U-PLEX plates for later use, keep each plate pouch and the desiccant that came with the plate. After the plates are incubated with the coating solution, wash them with MSD Wash Buffer or PBS-T, then return each plate to its original packaging with the desiccant, and seal. 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 Read Buffer T is at room temperature when added to a plate. Do not shake the plate after adding Read Buffer T. To improve inter-plate precision, keep time intervals consistent between adding Read Buffer T and reading the plate. Unless otherwise directed, read the plate as soon as possible after adding Read Buffer T. 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 v5-2018Apr 14

15 Typical Protocols A. Using MSD reagents Follow this protocol for R-PLEX and U-PLEX assays using MSD reagents. 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 (page 7). Prepare U-PLEX Plate The preparation of a U-PLEX plate involves coating the provided plate with Linker-coupled capture antibodies. A U-PLEX 4-Assay plate is shown below as an example, with four activated spots at locations 1, 3, 8, and 10. Assign each antibody to a unique Linker and record the antibody identity next to the assigned Linker, as shown below. Figure 5. A U-PLEX 4-Assay Plate, with recorded antibodies and assigned Linkers The protocol in this section describes the preparation of a multiplex coating solution for one 96-well plate. The volumes can be adjusted depending on the number of plates or wells, but the ratios of the reagents should remain the same. STEP 1: Create Individual Linker-Coupled Antibody Solutions A different Linker must be used for each unique biotinylated antibody. The number and layout of the active spots on the plate depends on the number of assays to be multiplexed. As an example, the steps to complete the coupling reactions for a 4-Assay plate are provided below. Couple each biotinylated capture antibody to a unique Linker and record the antibody identity next to the Linker number on the Spot Map (blank Spot Map is provided on page 32). Add 200 µl of each biotinylated antibody to 300 µl of the assigned Linker. Mix by vortexing. Incubate at room temperature for 30 minutes. Do not shake v5-2018Apr 15

16 Notes: Each Linker vial has a matching colored cap and label. To remove liquid from the cap, briefly centrifuge the Linker vial and open the cap gently. Open one Linker at a time and close its cap as soon as you are done using it. Take precautions to avoid reagent contamination. For studies using multiple plates of the same assay, it is recommended that the same Linker be coupled with the same antibody for the duration of the study. Add 200 µl of Stop Solution. Mix by vortexing. Incubate at room temperature for 30 minutes. Note: At the end of Step 1, each individual Linker-coupled antibody solution is at 10X the coating concentration and can be stored at 2-8 C. Do not store for more than 7 days. Adjust the volumes for multiple plates. The volumetric ratio of Linker: antibody: Stop Solution is 3:2:2. STEP 2: Prepare the Multiplex Coating Solution Combine 600 µl of each Linker-coupled antibody solution into a single tube and vortex. Up to 10 Linker-coupled antibodies can be pooled. Do not combine Linker-coupled antibody solutions that share the same Linker. When combining fewer than 10 antibodies, bring the solution up to 6 ml with Stop Solution. This will result in a final 1X concentration. Mix by vortexing. For example, for a 4-assay coating solution, add 3.6 ml of Stop Solution to the 2.4 ml of combined antibodies. Note: At the end of Step 2, the multiplex coating solution is at 1X and can be stored at 2-8 C. Do not store for more than 7 days. STEP 3: Coat the U-PLEX Plate Add 50 µl of multiplex coating solution to each well. Seal the plate with an adhesive plate seal and incubate at room temperature for 1 hour or overnight at 2-8 C. 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 and ready for use. Plates may be stored in the original pouch with desiccant and sealed, for up to 7 days at 2-8 C. The recommended volumes of Linker, biotinylated capture antibody, and Stop Solution for coating one or multiple U-PLEX plates are provided below. If using a partial plate (fewer than 96 wells), refer to Table 15. Table 12. Amount of each component required for U-PLEX coating solution per plate No. of Plate(s) Individual Linker ( µl) Individual Biotinylated Antibody (µl) Stop Solution (µl) , ,500 1,000 1,000 N 300 x N 200 x N 200 x N v5-2018Apr 16

17 Prepare Calibrator Standards MSD supplies vials with multi-analyte calibrator blend(s) covering the U-PLEX analytes and one vial of frozen individual calibrator(s) for each of the R-PLEX analytes on the plate. The frozen calibrators for R-PLEX are at 20-fold higher concentration than the top-of-the-curve concentration. You will receive one vial of each calibrator per plate. To prepare calibrator solutions for up to 4 replicates: 1) Prepare the frozen R-PLEX assay calibrator: a) Thaw the frozen R-PLEX assay calibrator on wet ice for at least 30 minutes and keep on ice. b) If only frozen R-PLEX calibrators are included in the kit, continue to Step 3. 2) Reconstitute the lyophilized U-PLEX calibrator(s): a) Add 125 µl of assay diluent to each of the lyophilized calibrator vials supplied. This will result in a 10X concentrated stock of the Calibrator, which will need to be diluted 10-fold (per the instructions in step 3) to generate the highest point in the standard curve (i.e., Calibrator Standard 1). b) Invert the reconstituted Calibrator at least 3 times. Do not vortex. Let the reconstituted solution equilibrate at room temperature for minutes and then vortex briefly. The Calibrator is now ready for use. Keep the dilutions at room temperature. 3) Prepare 8 Calibrator Standards (7 calibrator solutions plus a zero calibrator) for R-PLEX and/or U-PLEX calibrators: a) Prepare Calibrator Standard 1 by combining 15 µl of each R-PLEX 20X calibrator and/or 30 µl of each U-PLEX 10X calibrator (if applicable). Add assay diluent to bring up the volume to 300 µl. Mix by vortexing. b) For Calibrator Standard 2, add 50 µl of Calibrator Standard 1 to 150 µl of the assay diluent. Mix by vortexing. c) Repeat 4-fold serial dilutions 5 additional times to generate a total of 7 Calibrator Standards. Mix by vortexing between each serial dilution. d) Use assay diluent as Calibrator Standard 8 (zero Calibrator). Discard any unused, diluted calibrators. Note: For the lot-specific concentration of the analytes in U-PLEX Calibrators, refer to the COA received with your order and available at The values in the COA were prepared according to the U-PLEX Biomarker assay protocols. The protocol above is a modified reconstitution procedure that enables the combination of U-PLEX calibrators with R-PLEX calibrators. The COA provides the Calibrator concentrations for a 5-fold dilution; in this assay, the concentrations need to be adjusted since the U-PLEX Calibrator blend is a 10-fold dilution. For the R-PLEX analyte, refer to the datasheet supplied with the product for top-ofthe-curve concentration. Table 13. Serial dilutions to generate the standard curve Calibrator Standard # Tube # Source of Calibrator Volume of Reconstituted Calibrator (µl) Assay Diluent (µl) 1 1 Calibrator Standard 1 (top of curve) See above instructions Total volume (µl) 2 2 From tube From tube From tube From tube From tube From tube (zero Calibrator) v5-2018Apr 17

18 Figure 6. Dilution schema for preparation of Calibrator Standards for multiplex assays Dilute Samples Depending on the sample set under investigation, a dilution may be necessary. Assay diluent may be used for sample dilution. The dilution factor for the 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 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 each 100X detection antibody Antibody diluent to bring the final volume to 6 ml Prepare Read Buffer T (2X) MSD Read Buffer T (4X) is included in the U-PLEX Development Pack. The working solution is 2X. For one plate, combine: 10 ml of Read Buffer T (4X) 10 ml of deionized water Equilibrate the read buffer to room temperature before use. To avoid bubbles, do not vortex. You may keep excess diluted Read Buffer T in a tightly sealed container at room temperature for up to one month. Note: MSD GOLD Read Buffer can be substituted for MSD Read Buffer T. MSD GOLD Read Buffer is provided at the working concentration of the assay v5-2018Apr 18

19 B. Using your own reagents Follow this protocol when developing assays with your own reagents. Guidance on preparing calibrators and controls is provided in Appendix A. Coating with Antibodies Multiplexing assays on U-PLEX plates is achieved through specific capture material on each spot and coupling with a unique Linker. All capture antibodies (or other suitable capture reagents) should be biotinylated. Guidance on biotin conjugation is provided in Appendix A. When coating a multiplex plate, it is important to couple each individual biotin antibody to a unique Linker. The protocol in this section describes the preparation of multiplex coating solution for a single 96-well plate. The volumes can be adjusted depending on the number of plates or wells, but the ratios of the reagents should remain the same (Tables 14 and 15). You may adjust the capture antibody concentration and the final coating buffer concentration, but most assays do not benefit from increasing the capture antibody concentration. STEP 1: Create Individual U-PLEX-Coupled Antibody Solutions Couple individual biotinylated antibody (or other suitable capture reagent) to a unique Linker and record the antibody identity next to the Linker number on the Spot Map (a copy is provided on page 32). Dilute each biotinylated antibody to 10 µg/ml in coating diluent for a final volume of 200 µl per plate. The antibody solution should not contain free biotin. Coating diluents can be simple diluents using 0.5% bovine serum albumin in PBS or MSD Diluent 100. MSD offers a variety of diluents that could be used to dilute the capture antibody. 1% Blocker D-M, D-R, D-B and/or D-G could be added to the capture antibody diluent to reduce antibody-antibody non-specific interactions. Add 200 µl of each biotinylated antibody to 300 µl of the assigned Linker. A different Linker should be used for each unique biotinylated antibody. Vortex. Incubate at room temperature (RT) for 30 minutes. Refer to the U-PLEX plate Spot Map to determine which Linkers can be combined. For example, when coating 4 assays on a 4-Assay U-PLEX plate, the antibodies should be coupled to Linkers 1, 3, 8, and 10 respectively to match the Spot Map. Note: For long-term studies using multiple plates of the same assay, it is recommended that the same Linker be coupled with the same antibody for the duration of the study. Each Linker vial has a matching colored cap and label. Open one Linker at a time and close its cap before opening another Linker to avoid reagent contamination. Add 200 µl of Stop Solution. Vortex. Incubate at RT for 30 minutes. At the end of step 1, each individual U-PLEX-coupled antibody solution is at 10X the coating concentration and can be stored up to 7 days at 2-8 C. Adjust the volumes for multiple or partial plates as shown in Tables 14 and 15. The volumetric ratio of Linker:antibody:Stop Solution is 3:2: v5-2018Apr 19

20 STEP 2: Prepare Multiplex Coating Solution Combine 600 µl of each U-PLEX-coupled antibody solution into a single tube and vortex. Up to 10 U-PLEX-coupled antibodies can be pooled. Do not combine U-PLEX-coupled antibody solutions that share the same Linker. When combining fewer than 10 antibodies, bring the solution up to 6 ml with Stop Solution to result in a final 1X concentration. Vortex. For example, for a 4-assay coating solution, add 3.6 ml of Stop Solution to the 2.4 ml of combined antibodies. The multiplex coating solution can be stored up to 7 days at 2-8 C. STEP 3: Coat U-PLEX Plate Add 50 µl of multiplex coating solution to each well. Seal plate with an adhesive plate seal and incubate at RT for 1 hour or at 2-8 C for overnight. Shaking the plate during incubation is required. Wash plate 3 times with at least 150 µl/well of PBS-T (PBS plus 0.05% Tween-20) or MSD Wash Buffer. The plate is coated and ready for use. Plates may be stored in the original pouch with desiccant up to 7 days at 2-8 C. The recommended volumes of Linker, biotinylated antibody, and Stop Solution for coating one or multiple U-PLEX plates are provided below. The volumetric ratio of Linker:antibody:Stop Solution is 3:2:2. Table 14. Amount of each component needed for U-PLEX Coating Solution No. of Plate(s) Individual Linker (µl) Individual Biotinylated Antibody (µl) Stop Solution (µl) per reaction , ,500 1,000 1,000 N 300 x N 200 x N 200 x N The recommended volumes of Linker, biotinylated capture antibody, and Stop Solution for coating partial plates are provided below. Table 15. Amount of each component needed for U-PLEX Coating Solution (partial plate) No. of Wells Individual Linker (µl) Individual Biotinylated Capture Antibody (µl) Stop Solution per reaction (µl) Vol. to Pull from Each Reaction (µl) Coating with Non-Antibody Molecules Non-antibody molecules, such as biotinylated antigens and peptides, can also be immobilized on the U-PLEX plate. To optimize the coating concentrations, we recommend preparing the biotin molecules at 66, 33, and 16.7 nm. Calculation for converting from nm and µg/ml concentrations is available in Appendix A v5-2018Apr 20

21 Assay Protocol Note: Before beginning STEP 1, prepare the plate as described on page 15 (Using MSD reagents) or page 19 (Using your own reagents). 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 plate with an adhesive plate seal. Incubate at RT with shaking for 1 hour. STEP 2: Wash and Add Detection Antibody Solution Wash 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 plate with an adhesive plate seal. Incubate at RT with shaking for 1 hour. STEP 3: Wash and Read Wash plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 150 µl of 2X MSD Read Buffer T to each well. Analyze plate on an MSD instrument. Incubation in Read Buffer T 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 v5-2018Apr 21

22 Appendix A: Assay Optimization This section covers various topics to consider when developing and optimizing assays. Guidelines for Antibody Selection When the primary detection antibody is conjugated with SULFO-TAG label, it is important to confirm no cross-reactivity between capture and detection antibody. If the primary detection antibody is not conjugated with SULFO-TAG, use a SULFO-TAG conjugated secondary detection antibody that is raised against the host of the primary detection antibody. In such cases, the capture antibody and primary detection antibody should be raised in different host species. For example, if the capture antibody is raised in rabbit, choose a primary detection antibody raised in a different host species than rabbit (e.g., mouse). Since the capture antibody is always biotinylated, do not use a biotinylated detection antibody or Streptavidin-conjugated SULFO- TAG as a method for detection. Streptavidin-conjugated SULFO-TAG will cause high backgrounds, as it will bind to the biotin on the capture antibody. Coupling U-PLEX Linkers Coupling a U-PLEX Linker to a biotinylated capture reagent is simple add the capture reagent and the Linker together and vortex. There is no purification step necessary. When the capture reagent is an antibody, use the recommended 10 µg/ml. For nonantibodies, add 33 nm of the biotinylated reagent. Select the Linker number by looking at the Spot Map of the plate (page 32). When combining U-PLEX-coupled reagents, it is important that the coating solution contain a unique capture reagent for each Linker. Antibody Selection Sensitivity, specificity, and format of the assay are affected by selection of a capture antibody and a detection antibody, each of which must recognize a different non-overlapping epitope. U-PLEX provides an excellent platform for unbiased selection of antibodies and for determining the best performing antibody pairs. The most comprehensive way to identify antibody pairs is to test each combination of antibody as the capture antibody and as the detection antibody. The biotinylated capture antibodies can be coupled to different U-PLEX Linkers and up to 10 antibodies can be coated in a single well. Each detection antibody can be tested in its own well to provide up to 10 capture-detection antibody combinations per well. Antibody pairs can be tested in high throughput and selected based on a variety of different analyses including positive signal, signal to background, or change in treatment condition. For many assays, it is preferable to have both the capture antibody and detection antibody be monoclonal antibodies, each recognizing a unique epitope. Monoclonal antibodies are typically easier to reproduce from lot to lot and can be produced in large quantity leading to increased longevity of the assay. Selection of two monoclonal antibodies may not be possible because of reagent availability or desired sensitivity achieved with a polyclonal antibody. When both a monoclonal antibody and a polyclonal antibody are used, it is preferable to use the monoclonal antibody as the capture antibody and the polyclonal antibody as the detection antibody. The advantage of using a polyclonal antibody is that it may contain multiple antibodies that recognize different epitopes, leading to higher avidity. When a polyclonal antibody is not affinity purified, it contains non-specific antibodies that could lead to nonspecificity issues or reduced assay performance. An affinity purified polyclonal antibody has greater specificity than one purified by Protein A or G, but it may still exhibit lot-to-lot variability because each lot may be a different mixture of antibodies. When using a v5-2018Apr 22

23 polyclonal antibody as a capture antibody, it could contain a population of antibodies that share or block the detection antibody epitope, leading to reduced signals or sensitivity. Antibody Conjugation Consistent conjugation of the capture antibody and detection antibody are important for good assay performance. Optimization of the conjugation ratios is not necessary for assay development, but it could improve assay performance in certain situations. At least one biotin must be present on the capture antibody for it to be coupled to the U-PLEX Linker. The signal from an assay is proportional to the number of SULFO-TAG molecules conjugated to the detection protein. Over-conjugation of capture and detection antibodies can lead to reduced performance. We recommend, below, typical challenge ratios for biotinylation and SULFO- TAG conjugation that should work for most assays. SULFO-TAG Conjugation Ratio A 20:1 challenge ratio is recommended for SULFO-TAG conjugation of antibodies where high sensitivity is required. This challenge ratio leads to a typical conjugation ratio of 10:1. For immunogenicity assays, however, a 10:1 challenge ratio is recommended to reduce masking of the number of epitopes from being blocked. Smaller proteins (<50kDa) are often conjugated with a 5:1 challenge ratio since they have fewer sites for conjugation. The best practice is to purify the SULFO-TAG conjugated antibody to remove the unconjugated SULFO-TAG NHS-ESTER. However, unconjugated SULFO-TAG NHS-ESTER may not degrade the performance of the assay because it is washed away with the detection antibody, and ECL signal is generated only by SULFO-TAG that is near the electrode surface in the presence of Read Buffer when the instrument excites the well. To simplify antibody selection, it may be faster to test with non-purified SULFO-TAG conjugated antibody during antibody screening. Optimization of the SULFO-TAG challenge ratio may be necessary when high backgrounds are not reduced through simple diluent optimization. Lowering the challenge ratio should lower the background from the detection antibody and will likely reduce the assay sensitivity. To increase sensitivity, the challenge ratio could be increased, but remember that the performance will increase only to a certain point and then will start to degrade. The SULFO-TAG conjugation protocol is found in the the MSD GOLD SULFO-TAG Conjugation Quick Guide, available at Biotin Conjugation Ratio The recommended biotin challenge ratio for antibodies is 10 biotins to 1 capture antibody. This challenge ratio will lead to conjugation of an average of 2-4 biotins per antibody. In a study of 20 antibodies, we found that increasing the challenge ratio from 5:1 to 20:1 did not cause an increase in performance beyond the 10:1 challenge ratio. For a small sub-set of antibodies, the 10:1 challenge ratio resulted in a two-fold reduction in performance relative to the 5:1 challenge ratio. For best performance, it is important to purify the biotinylated antibody after conjugation. Unconjugated biotin in the capture antibody solution will interfere with the coupling step with the U-PLEX Linker. The graph at right shows that with 5% biotin in the solution, the performance of the assay is suppressed by 10%. The biotin conjugation protocol is found in the Biotin Conjugation Quick Guide, available at % Signal Normalized to 0 Free Biotin % of Free Biotin in Antibody Solution v5-2018Apr 23

24 Antibody Concentration Optimization Choosing the appropriate capture antibody and detection antibody concentrations is the first step in assay optimization. Antibody concentrations can be optimized to improve sensitivity, reduce background signals, and reduce reagent usage. Capture Antibody Concentration The optimal antibody concentration for the antibody-linker coupling step is 10 µg/ml. Assays tested during development of the U-PLEX platform did not benefit from using higher antibody concentrations. If made per the protocol, the final coating solution is 1X; add 50 µl of coating solution to each well. It is not recommended to increase the multiplex coating solution concentration beyond 1X because the Linker amount in the solution will exceed the guaranteed binding capacity of the plate. At these recommended concentrations, 2 µg of capture antibody is required per 96-well plate. To simplify assay development, use the recommended coating conditions and if optimization is required, titrate the coating solution down. Converting nm Biotinylated non-antibody molecules to µg/ml Concentration The following formula can be used to convert nm biotinylated protein or peptide to µg/ml concentration. µg/ml protein = UnM concentration of protein x molecular weight of protein (Da) 1,000,000 Detection Antibody Concentration Titration of the detection antibody should be done for each specific reagent to determine the best detection antibody concentration and typically ranges from 0.1 µg/ml up to 3 µg/ml. To reduce assay variability between runs and preparations, optimize the detection antibody concentration to minimize changes in signal. For most high affinity antibodies, the optimal detection antibody concentration will be less than 0.5 µg/ml. When developing a multiplexed assay, the best practice is to test detection antibodies individually to determine the impact of detection antibody concentration on the background signals on all capture antibodies. While antibody concentrations can be adjusted to reduce high backgrounds, reducing the number of SULFO-TAG labels on the detection antibody can have the same effect without altering the binding kinetics. The detection antibody titrations for three different assays are shown below. We recommend that you test at least 3 to 4 detection antibody concentrations to determine the appropriate final antibody concentration. Consider both the signal and background when choosing a final antibody concentration. One practice may be to select an antibody concentration above the midpoint of the curve Signal Signal Signal IL-1β Detection Antibody Concentration (µg/ml) IL-8 Detection Antibody Concentration (µg/ml) TNF-α Detection Antibody Concentration (µg/ml) v5-2018Apr 24

25 Calibration Curve An ideal calibrator should be representative of the native protein; select either a purified native protein or a recombinant protein that resembles the native form whenever possible. The antibodies may preferentially recognize the calibrator or standard over the native protein, especially if the calibrator was used to generate the antibody. Recombinant proteins or standards may not be available for all assays. In these cases, such as with intracellular signaling assays, an appropriate cell model may be developed to generate a native form of the protein. Select a simple diluent, such as Diluent 100, containing a carrier protein to be used as the calibrator diluent. If the samples to be tested are in a complex matrix, serum-containing diluents may be needed to match the performance of the calibrator in the diluent to analyte in the matrix to be tested. Choosing the right concentrations for the calibration curve is important for maximizing the dynamic range of the assay. Assay signals on MSD instruments can have a background as low as 50 counts and can saturate above 1,000,000 ECL counts. A good place to start optimization for a biomarker assay is with the top calibrator at 10,000 times the expected sensitivity of the assay. For example, if the expected sensitivity is 1 pg/ml, the top of the curve should be 10 ng/ml. A four-fold dilution between calibrator points will provide a calibrator range of 16,384 fold between the first and 7th calibrator points. A blank should be run as part of the assay optimization. The top calibrator concentration can be adjusted to generate a signal near or below 1,000,000 counts. When multiplexing different assays, different top calibrator concentrations may be required to maximize dynamic range. A larger dilution factor or more calibrator points may be needed to cover the entire assay range. For intracellular signaling markers, if recombinant calibrators are not available, an appropriate cell model may be developed to be used for positive and negative controls in the assay. Positive and negative cell lysates may be used neat or after dilution. A good starting concentration is 20 µg/well lysate with 2-fold dilutions. For cell lysates, protease and phosphatase inhibitors may be required in the extraction buffer to preserve the integrity of the analyte. Avoid reagents that could denature the capture antibody (general guidelines are: ionic detergents such as SDS should be <0.1%; reducing agents such as DTT should be <1 mm in the calibrator when added to the well). If high concentrations of potentially denaturing agents are required for extraction, the calibrator should be diluted in a suitable buffer lacking denaturing agent before adding to the antibody coated plate. Prepare Calibrators and Controls Secreted biomarkers For secreted biomarker assays, a calibrator or a recombinant protein that is representative of the native protein can be used for the calibration curve. A good starting concentration is 10 ng/ml for the high calibrator and ng/ml for the low calibrator concentration. We recommend testing an 8-point titration curve for biomarker assays and optimizing the calibrator diluent if required. Denaturing agents should be avoided or kept to a minimum (SDS should be <0.1%, reducing agents such as DTT should be <1 mm in the sample when added to the well). Keep diluted samples on ice until use. Intracellular signaling markers If recombinant calibrators are not available, an appropriate cell model may be developed to be used for positive and negative controls in the assay. MSD offers cell lysate controls for a range of phosphoprotein and signaling pathway kits. Further details on cell lysate controls are available at v5-2018Apr 25

26 Positive and negative cell lysates may be used neat or after dilution. A good starting concentration is 20 µg/well lysate with 2-fold dilutions. MSD plates are compatible with most sample matrices. Avoid reagents that could denature the capture antibody (general guidelines are: ionic detergents such as SDS should be <0.1%; reducing agents such as DTT should be <1 mm in the sample when added to the well). If high concentrations of potentially denaturing agents are required for extraction, the sample should be diluted in a suitable buffer lacking denaturing agent before adding to the antibody coated plate. For cell lysates, protease and phosphatase inhibitors may be required in the extraction buffer to preserve the integrity of the analyte. Keep diluted samples on ice until use v5-2018Apr 26

27 Appendix B: Assay Performance U-PLEX is an assay development tool that enables you to coat multiplex plates with ease and flexibility. It is an excellent platform for creating assays of your choice, screening a large number of antibodies, or testing the feasibility of multiplexing assays together. You may need to optimize your assays once the capture antibodies are coated on the U-PLEX plate. The assay performance and quality will vary depending on a number of factors, including antibody affinity and concentration, diluent selection, incubation time, and the combination of multiplexed assays. To evaluate the quality of the U-PLEX platform, we tested over 175 assays that were already optimized and validated, the majority of which share the same antibodies and reagents as the V-PLEX product line. These assays were tested for sensitivity, dynamic range, precision, and sample quantitation on U-PLEX. Typical Calibration Curves Representative calibration curves from 2 sets of multiplexed assays on U-PLEX plates are presented below. Assays performed on U-PLEX typically show 3-4 log dynamic range. Wide dynamic range allows simultaneous quantitation of normal and disease/stimulated samples with the same sample dilution. Sensitivity For each assay, we measured the lower limit of detection (LLOD), which is the calculated concentration corresponding to the signal 2.5 standard deviations above the background. The median LLODs on U-PLEX were evaluated based on at least 17 plates across multiple independent runs. Representative LLODs are presented below. LLODs may vary depending on the antibodies used in developing the assays. LLOD (pg/ml) Assays IFN-γ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNF-α U-PLEX LLOD (pg/ml) Assays Eotaxin IP-10 MCP-1 MCP-4 MDC MIP-α MIP-β TARC U-PLEX v5-2018Apr 27

28 Reproducibility We evaluated the reproducibility of the U-PLEX platform by measuring the intra-plate, inter-plate, and inter-lot CVs of calibrators and controls. Representative data from 8 assays are shown below. The same assays were tested across 3 lots of U-PLEX plates and 3 lots of Linkers. Calibrators within the quantifiable range of each assay were evaluated. The average intra-plate and inter-plate CVs for both the calibrator signals and the back-fitted calculated concentrations for most assays were less than 10% in all the lots tested (data not shown). Reproducibility of controls at 3 different levels is shown below. The average intra-plate and inter-plate CVs for most assays were below 7%. Lot 1 (N = 9 Plates) Lot 2 (N = 6 Plates) Lot 3 (N = 9 Plates) Inter-Lot Eotaxin MIP-1β TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Control Average Conc. (pg/ml) Average Intra- Plate %CV Inter- Plate %CV Average Conc. (pg/ml) Average Intra- Plate %CV Inter- Plate %CV Average Conc. (pg/ml) Average Intra- Plate %CV Inter- Plate %CV Average Inter-lot Conc. High Mid Low High Mid Low High Mid Low High 1, , , , Mid , Low High Mid Low High Mid Low High 3, , , , Mid 2, , , , Low High Mid Low Interlot %CV Sample Quantitation Forty human serum and 40 EDTA plasma samples were tested on both U-PLEX and V-PLEX using the same antibodies, calibrators, and diluents. Overall, there is high correlation between the V-PLEX assays and the same assays performed on U-PLEX as shown in the table. Sample Type Statistic Eotaxin MIP-1β TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Serum EDTA Plasma r 2 Value Slope r 2 Value Slope In summary, U-PLEX is an excellent platform for creating personalized multiplex assays. The U-PLEX platform preserves the characteristics of a high-quality assay, including low detection limit, wide dynamic range, and high precision v5-2018Apr 28

29 Appendix C: Assay/Antibody Diluent Combinations Below is additional information on the diluent combinations for serum and plasma samples 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 Human Assay Diluent 2 Antibody Diluent 3 Alpha-amylase CA CA CA50 20 CD5* Neat CD27 Neat CTLA-4 2 E-Cadherin 20 Galectin-3 50 Granzyme A 2 Granzyme B 2 LAG3 2 MET (soluble) 20 MIG 4 NT-proBNP 50 Osteonectin 20 Osteoprotegerin 2 PD1 2 PD-L1 2 PD-L2 2 Resistin 16 TLR1 2 TNF-RII 10 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 PRDX-6 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 SCFR/Kit 50 Tie-2 2 VEGF-D 2 Assay Diluent 7 Antibody Diluent 22 Troponin T (cardiac) 20 Assay Diluent 7 Antibody Diluent 37 Annexin A1 4 CEA 4 Osteopontin 4 IL1RL1/ST2 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 3 FABP3/H-FABP 2 Assay Diluent 12 Antibody Diluent 11 PYY (active)* 2 Glucagon 2 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 HGF 2 HSP70 2 MMP-7 2 OX40 2 OX40L 2 Pentraxin 3 2 RAGE (soluble) 2 S100A8/MRP8 2 S100A12 2 Serpin A1 4,000 Serpin A12/Vaspin 2 Tenascin C 100 TNFRSF10C 20 U-PAR 2 vwf 1,000 Assay Diluent 13 Antibody Diluent 12 Complement factor D 100 C-Peptide Neat FGF-21 12,000 FGF-23 4 FSH Neat GIP (active)* 2 GIP (inactive)* 2 GIP (total)* 2 Insulin Neat Leptin Neat LH Neat Prolactin 10 Assay Diluent 37 Antibody Diluent 37 AGP 300,000 ApoA1 300,000 ApoE 4,000 Complement C3 300,000 EGF 500 Haptoglobin 300,000 HAVCR1/KIM-1 * 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 Ghrelin (total) 2 Gelsolin 100 PP 8 TfR-1 (soluble) 100 TIMP 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. *Please review datasheet. Diluent combinations for human analytes are shown above; see next page for mouse, NHP, and rat analytes v5-2018Apr 29

30 Mouse Assay Diluent 11 Antibody Diluent 11 Resistin 20 Assay Diluent 100 Antibody Diluent 17 Leptin Neat Assay Diluent 100 Antibody Diluent 100 Adiponectin 1,000 Mouse/Rat Assay Diluent 13 Antibody Diluent 12 C-Peptide Neat NHP Assay Diluent 2 Antibody Diluent 3 Alpha-amylase 2A 5,000 *Please review datasheet. Rat Assay Diluent 6 Antibody Diluent 100 Leptin Neat Assay Diluent 7 Antibody Diluent 100 NGAL/LCN2 100 Assay Diluent 11 Antibody Diluent 5 TIMP-1 20 Assay Diluent 29 Antibody Diluent 100 HAVCR1/KIM-1 * Osteopontin * Assay Diluent 42 Antibody Diluent 40 EPO Neat GM-CSF 2 IL-1α 4 IL-1β 2 IL-2 4 IL-4 2 IL-5 4 IL-6 2 IL-10 Neat IL-13 8 KC/GRO 4 MCP-1 Neat MIP-3α Neat TNF-α 2 VEGF-A Neat 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 v5-2018Apr 30

31 Summary Protocol for Multiplex Assays U-PLEX Plate Preparation STEP 1: Create Individual U-PLEX-Coupled Antibody Solutions Couple an individual biotinylated antibody to a unique Linker and record the antibody identity next to the Linker number on the Spot Map on the following page. If using your own antibodies, dilute each biotinylated antibody to 10 µg/ml in coating diluent for a final volume of 200 µl per plate. Add 200 µl of each biotinylated antibody to 300 µl of the assigned Linker. Refer to the U-PLEX plate Spot Map to determine which Linkers can be combined. A different Linker should be used for each unique biotinylated antibody. Vortex. Incubate at room temperature (RT) for 30 minutes. Add 200 µl of Stop Solution. Vortex. Incubate at RT for 30 minutes. STEP 2: Prepare Multiplex Coating Solution Combine 600 µl of each U-PLEX-coupled antibody solution into a single tube and vortex. Up to 10 U-PLEX coupled antibodies can be pooled. Do not combine U-PLEX coupled antibody solutions that share the same Linker. When combining fewer than 10 antibodies, bring the solution up to 6 ml with Stop Solution to result in a final 1X concentration. Vortex. STEP 3: Coat U-PLEX Plate Add 50 µl of multiplex coating solution to each well. Seal plate with an adhesive plate seal and incubate at RT for 1 hour or overnight. Shaking the plate during incubation is required. Wash plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Plate is coated and ready for use. Summary Assay Protocol Note: Before beginning STEP 1, prepare the U-PLEX plate as described above. 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 RT with shaking for 1 hour. STEP 2: Wash and Add Detection Antibody Solution Wash 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 RT with shaking for 1 hour. STEP 3: Wash and Read Wash plate 3 times with at least 150 µl/well of 1X MSD Wash Buffer or PBS-T. Add 150 µl of 2X MSD Read Buffer T (or MSD GOLD Read Buffer) to each well. Analyze plate on an MSD instrument. Incubation in Read Buffer T is not required before reading the plate v5-2018Apr 31

32 Spot Map Map your assay spot location by writing the name of each analyte next to its spot number v5-2018Apr 32