U-PLEX Biomarker Group 2 (Human, Mouse, and NHP)

Transcription

1 U-PLEX Biomarker Group 2 (Human, Mouse, and NHP) Individual Assays FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES v1-2007Mar

2 MSD U-PLEX Platform U-PLEX Biomarker Group 2 (Human, Mouse, and NHP) Individual Assays For use with serum, EDTA plasma, and cell culture supernatants. Catalog numbers for U-PLEX Biomarker Group 2 individual assay packs are provided in Table 5 on page 13. 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 17Twww.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, 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), 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 Meso Scale Diagnostics, LLC. All rights reserved v1-2017Oct 2

3 Table of Contents Introduction... 4 Principle of the Assay... 5 Components... 6 Additional Materials and Equipment... 8 Safety... 8 Best Practices... 9 Reagent Preparation Assay Protocol Assay Performance Appendix Summary Protocol Plate Diagram Contact Information MSD Customer Service Phone: Fax: CustomerService@mesoscale.com MSD Scientific Support Phone: Fax: Attn: Scientific Support ScientificSupport@mesoscale.com v1-2017Oct 3

4 Introduction Traditional immunoassays, such as ELISAs, provide assay choice but sacrifice sensitivity and read time. The MSD U-PLEX platform combines high sensitivity and a rapid read time (less than 2 minutes) with the flexibility to easily design and build custom assays and efficiently transition from individual to multiplex assays. Individual assays, utilizing U-PLEX Antibody Sets with MSD GOLD Small Spot Streptavidin Plates, have low detection limits, provide up to 5 logs of linear dynamic range, and use minimal sample volume. The U-PLEX Biomarker Group 2 contains three analytes (Table 1) that are important in many biological processes. These assays can detect secreted biomarkers in a variety of body fluids and cell culture supernatants where over- or under-expression may indicate a shift in the biological equilibrium. A representative data set for each of the assays in U-PLEX Biomarker Group 2 is presented in the product-specific datasheets available at Table 1. Assays included in U-PLEX Biomarker Group 2 Assay Human Mouse NHP (Cat. #) (Cat. #) (Cat. #) TGF-β1 K151XWK K152XWK K156XWK TGF-β2 K151XUK K152XUK K156XUK TGF-β3 K151XVK K152XVK K156XVK v1-2017Oct 4

5 Principle of the Assay Individual assays are supplied on MSD GOLD Small Spot Streptavidin Plates (Figure 1). These plates provide high sensitivity, consistent performance, and excellent inter- and intra-lot uniformity. Each individual assay is supplied with a biotinylated capture antibody that binds to streptavidin on the plate surface. Analytes 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 sandwich immunoassay. 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 each analyte in the sample. Figure 1. U-PLEX individual assay on a MSD GOLD Small Spot Streptavidin Plate v1-2017Oct 5

6 Components The following tables list the components provided with U-PLEX Biomarker Group 2 individual assays including Antibody Set, Calibrator, and general reagents. You will only receive components relevant to the assays that you order. Reagents Supplied With All U-PLEX Individual Assays Table 2. Plates and reagents that are supplied with all U-PLEX Biomarker Group 2 individual assays Reagent Storage Catalog # Size MSD GOLD 96-Well Small Spot Streptavidin SECTOR Plate Quantity Supplied 1 Plate 5 Plates 25 Plates Description 2 8 C L45SA-1 1-spot 1 plate 5 plates 25 plates 96-well plate, foil sealed, with desiccant Diluent C R50AA-4 50 ml 1 bottle 1 bottle 5 bottles Diluent for biotinylated capture antibody Read Buffer T (4X)* RT R92TC-3 50 ml 1 bottle 1 bottle 5 bottles Diluent 43 Diluent 3 Diluent 41 Diluent C -10 C -10 C -10 C Human and NHP Assays Buffer to catalyze the electrochemiluminescence reaction. Dilute to 2X and use at room temperature. R50AG-1 10 ml 1 bottle Diluent for samples and Calibrators; contains serum, blockers, and preservatives. R50AG-2 50 ml 1 bottle 5 bottles R50AP-1 8 ml 1 bottle Diluent for detection antibody; contains R50AP-2 40 ml 1 bottle 5 bottles protein, blockers, and preservatives. Mouse Assays R50AH-1 10 ml 1 bottle Diluent for samples and Calibrators; contains serum, blockers, and preservatives. R50AH-2 50 ml 1 bottle 5 bottles R50AI-3 8 ml 1 bottle Diluent for detection antibody; contains protein, blockers, and preservatives. R50AI-4 40 ml 1 bottle 5 bottles *MSD GOLD Read Buffer (Cat. # R92TG-2) can be substituted for Read Buffer T. MSD GOLD Read Buffer is provided at the working concentration of the assay v1-2017Oct 6

7 Assay-Specific Reagents U-PLEX Antibody Set Based upon the analyte selected, you will receive a U-PLEX Antibody Set containing a biotinylated capture antibody and SULFO-TAG conjugated detection antibody. The biotinylated capture antibody is provided at a ready-to-use concentration, and the SULFO-TAG conjugated detection antibody is provided at a 100X concentration. A complete list of all Antibody Sets available for U-PLEX Biomarker Group 2 and their respective catalog numbers is provided in the Appendix (Table 6). Table 3. Contents of U-PLEX Antibody Set Name Storage Size Quantity Supplied 1 Plate 5 Plates 25 Plates Description U-PLEX Group 2 Analyte-Specific Antibody Set 2 8 C 1-Plate 1 Set containing biotinylated capture antibody and SULFO-TAG 5-Plate 1 5 conjugated detection antibody. Calibrators Calibrators are multi-analyte blends, each containing multiple recombinant human proteins lyophilized in a buffered diluent. Individual analyte concentrations are provided in the lot-specific certificates of analysis (COA). The following multi-analyte Calibrator is provided with all Group 2 assays. Table 4. Calibrator 11 is used for all U-PLEX Biomarker Group 2 assays Name Storage Catalog # Size Quantity Supplied 1 Plate 5 Plates 25 Plates Analytes Calibrator C C vial 1 vial 5 vials 25 vials TGF-β1, TGF-β2, TGF-β v1-2017Oct 7

8 Additional Materials and Equipment Appropriately sized tubes for reagent preparation Polypropylene microcentrifuge tubes for preparing dilutions Liquid handling equipment suitable for dispensing 10 to 225 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 MSD Wash Buffer (20X, 100 ml, catalog # R61AA-1) or Phosphate-buffered saline (PBS) plus 0.05% Tween-20 (PBS-T) for plate washing This size of MSD Wash Buffer is sufficient for washing four plates manually or for washing two plates with an automated plate washer Prepare a 1X working solution. For one plate, combine 15 ml of MSD Wash Buffer (20X) with 285 ml of deionized water Adhesive plate seals Deionized water Vortex mixer 1M HCl 1.2M NaOH in 0.5M HEPES ph paper to confirm neutralization of samples (optional) 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 v1-2017Oct 8

9 Best Practices Bring frozen diluent to room temperature in a C water bath. If you are running multiple assays, avoid cross-contamination between antibodies by following the techniques below: o Open one capture antibody vial at a time. Close the cap after use. o Use filtered pipette tips. o Use a fresh pipette tip after each reagent addition. 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 T 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 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. The uncoated wells of a partially used plate may be sealed and stored up to 30 days at 2 8 C in the original foil pouch with desiccant. You may adjust volumes proportionally when preparing reagents v1-2017Oct 9

10 Reagent Preparation Bring all reagents to room temperature and refer to the Best Practices section (page 9) before beginning the protocol. Important: Upon first thaw, aliquot assay Diluents into suitable volumes before refreezing. To prepare MSD Wash Buffer and other supplemental reagents, please refer to the Additional Materials and Equipment section (page 8). Coat Plate Add 200 µl of biotinylated capture antibody to 3.3 ml of Diluent 100. Mix by vortexing. Add 25 µl of the above solution to each well of the provided MSD GOLD Small Spot Streptavidin Plate. Seal the plate with an adhesive plate seal and incubate with shaking at room temperature 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 (PBS plus 0.05% Tween-20). The plate is now coated and ready for use. Prepare Calibrator Standards The following instructions will enable you to prepare seven Calibrator Standard solutions plus a zero Calibrator Standard for up to six replicates. Bring the Calibrator vial to room temperature. Reconstitute the Calibrator by adding 250 µl of Diluent 43 (Human or NHP) or Diluent 41 (Mouse) to the glass vial. Do not acid-treat the Calibrator. 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. This will result in a 5X concentrated stock of the Calibrator. Dilute the 5X Calibrator stock solution. Dilute 50 µl of Calibrator stock with 200 µl of Diluent 43 (Human and NHP) or Diluent 41 (Mouse) to generate the highest point in the standard curve (i.e., Calibrator Standard 1). Calibrator Standard 1 is now ready for use. Keep dilutions at room temperature. Note: We recommend that reconstituted Calibrators are used immediately. If storage is necessary, divide into 60 µl aliquots and store immediately at -70 C. Prepare Calibrator Standard 2 by adding 75 µl of Calibrator Standard 1 to 225 µl of Diluent 43 (Human or NHP) or Diluent 41 (Mouse). Mix by vortexing. Repeat 4-fold serial dilutions 5 additional times to generate a total of 7 Calibrator Standards. Mix by vortexing between each serial dilution. Use Diluent 43 (Human and NHP) or Diluent 41 (Mouse) as Calibrator Standard 8 (zero Calibrator/blank). Note: For the lot-specific concentration of Calibrators in the blend, refer to the COA supplied with the assay pack. You can also find a copy of the COA at v1-2017Oct 10

11 Figure 2. Dilution schema for preparation of Calibrator Standards for U-PLEX Biomarker Group 2 individual assays Prepare Samples Note: TGF-β samples typically require an acid treatment for activation. Prepare TGF-β samples as follows: Add 20 µl of 1M HCl per 100 µl of neat sample volume. Vortex briefly. Incubate sample for 10 min at room temperature. Neutralize the sample by adding 14 µl of 1.2M NaOH in 0.5M HEPES per 100 µl of sample volume. Vortex briefly. Samples are ready to use. Use immediately. Depending on the assay and sample set under investigation, a dilution may be necessary. Diluent 43 (for Human and NHP) or Diluent 41 (for Mouse) may be used for sample dilution. The dilution factor for the given sample type needs to be optimized. 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 Diluent 3 (Human and NHP) or Diluent 45 (Mouse) to bring the final volume to 6 ml Prepare Read Buffer T MSD provides Read Buffer T as a 4X stock solution. The working solution is 2X. For one plate, combine: 10 ml of Read Buffer T (4X) 10 ml of deionized water 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 (Cat. # R92TG-2) can be substituted for Read Buffer T. MSD GOLD Read Buffer is provided at the working concentration of the assay v1-2017Oct 11

12 Assay Protocol Note: Follow Reagent Preparation before beginning this assay protocol. STEP 1: Add Samples and Calibrators Add 25 µl of Diluent 43 (Human and NHP) or Diluent 41 (Mouse) 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 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 2X Read Buffer T to each well. Analyze the 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. Assay Performance A representative data set for each assay is presented in the product-specific datasheets available at The data represent performance of the assay tested in multiplex 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 v1-2017Oct 12

13 Appendix U-PLEX Assays Assay packs include Antibody Sets, plates, diluents, Calibrators, and Read Buffer T. Table 5. Catalog numbers of U-PLEX Biomarker Group 2 individual assay packs Product U-PLEX TGF-β1 Assay (hu) U-PLEX TGF-β2 Assay (hu) U-PLEX TGF-β3 Assay (hu) U-PLEX TGF-β1 Assay (ms) U-PLEX TGF-β2 Assay (ms) U-PLEX TGF-β3 Assay (ms) U-PLEX TGF-β1 Assay (NHP) U-PLEX TGF-β2 Assay (NHP) U-PLEX TGF-β3 Assay (NHP) Catalog Numbers (-1/-5/-25 Plate Size) K151XWK-1/-2/-4 K151XUK-1/-2/-4 K151XVK-1/-2/-4 K152XWK-1/-2/-4 K152XUK-1/-2/-4 K152XVK-1/-2/-4 K156XWK-1/-2/-4 K156XUK-1/-2/-4 K156XVK-1/-2/-4 U-PLEX Antibody Sets Antibody Sets include a biotinylated capture antibody and SULFO-TAG conjugated detection antibody. Table 6. Catalog numbers of Antibody Sets available for U-PLEX Biomarker Group 2 Product U-PLEX TGF-β1 Antibody Set U-PLEX TGF-β2 Antibody Set U-PLEX TGF-β3 Antibody Set Catalog Numbers (-1/-5 Plate Size) B20XW-2/-3 B20XU-2/-3 B20XV-2/ v1-2017Oct 13

14 Summary Protocol Coat Plate Add 200 µl of biotinylated capture antibody to 3.3 ml of Diluent 100. Mix by vortexing. Add 25 µl of the above solution to each well of the provided MSD GOLD Small Spot Streptavidin Plate. Seal the plate with an adhesive plate seal and shake for 1 hour at room temperature. Alternatively, you can also shake the plate overnight while incubating at 2-8 C. 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. Assay Protocol STEP 1: Add Samples and Calibrators Add 25 µl of Diluent 43 (Diluent 41 for Mouse assays) 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 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 Wash Buffer or PBS-T. Add 150 µl of 2X Read Buffer T to each well. Analyze the plate on an MSD instrument. Incubation in Read Buffer T is not required before reading the plate v1-2017Oct 14

15 Plate Diagram Figure 3. Plate diagram. A similar plate layout can be created in Excel and easily imported into DISCOVERY WORKBENCH software v1-2017Oct 15