Jan 30, 2025
U-PLEX® Metabolic Group 1 (mouse) Multiplex Assays
- Alexandria White1,
- Jianjun Chang1
- 1Emory University
Collection Citation: Alexandria White, Jianjun Chang 2025. U-PLEX® Metabolic Group 1 (mouse) Multiplex Assays. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8dq5rg2w/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: December 19, 2024
Last Modified: January 30, 2025
Collection Integer ID: 119304
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Against Parkinson's (ASAP)
Grant ID: ASAP-020527
Abstract
The U-PLEX platform allows the creation of custom multiplex assays for any combination of analytes in the same Group.
This product insert is for U-PLEX Combos and multiplex assays that contain any number of the 58 assays in the U-PLEX Metabolic Group 1 (mouse). Using open spots, custom combinations can include R-PLEX® Antibody Sets or your antibodies for other analytes.
Representative data for each U-PLEX assay is presented in the product-specific datasheets available at the www.mesoscale.com® website. The performance of MSD assays may vary when tested in combination. The data presented in the datasheets do not represent the product specifications
Guidelines
Principle of the Assay
Biotinylated capture antibodies are coupled to U-PLEX Linkers, which self-assemble onto unique spots on the U-PLEX plate. 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 (Figure 1). 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) and provides a quantitative measure of each analyte in the sample.
Figure 1. U-PLEX multiplex immunoassay on a U-PLEX 96-well 10-Assay Plate. U-PLEX 384-well 4-Assay plates are similar.
Spot Maps
Figure 5. Spot maps. (top) 96-well plate, (bottom) 384-well plate.
Materials
Components
Tables 1, 2, and 3 list the components provided with multiplex U-PLEX Metabolic Group 1 (mouse) Assays. You will only receive components relevant to the assays that you order.
| A | B | C | D | E | F | G | H | |
| Reagent | Storage | Catalog No. | Size | Quantity Supplied | Description | |||
| 1 Plate | 5 Plates | 25 Plates | ||||||
| Diluent 13 | ≤-10 °C | R56BB-4 | 10 mL | 1 bottle | — | — | Diluent for samples and calibrators | |
| R56BB-3 | 50 mL | — | 1 bottle | 5 bottles | ||||
| Diluent 11 | ≤-10 °C | R55BA-5 | 10 mL | 1 bottle | — | — | Diluent for detection antibody | |
| R55BA-3 | 50 mL | — | 1 bottle | 5 bottles | ||||
| Aprotinin (200,000 KIU/mL) | 2–8 °C | R93AD-1 | 50 µL | 1 vial | — | — | 200X concentrated solution | |
| R93AD-2 | 250 µL | — | 1 vial | 5 vials | ||||
| Stop Solution | 2–8 °C | R50AO-1 | 40 mL | 1 bottle | 1 bottle | 5 bottles | Biotin-containing buffer to stop Linker-antibody coupling reaction | |
| MSD GOLD Read Buffer B | RT | R60AM-1 | 18 mL | 1 bottle | — | — | Buffer to catalyze the electrochemiluminescent reaction | |
| R60AM-2 | 90 mL | — | 1 bottle | 5 bottles | ||||
Table 1. Reagents that are supplied with all U-PLEX Metabolic Group 1 (mouse) 96-well Assays
RT = room temperature
Dash (—) = not applicable
Assay-Specific Reagents
U-PLEX plates are provided in a sealed foil pouch with desiccant. The spots correspond to unique U-PLEX Linkers. The number and layout of the active spots on the plate depend on the plate well density (96 vs 384) and the number of assays to be multiplexed (Figure 2). For example, if 4 assays are to be multiplexed, either a U-PLEX 96-well or 384-well 4-Assay Plate will be provided.
U-PLEX Plates
U-PLEX multiplex assays include plates that are specific to the assay with which they ship. Do not separate plates from the other refrigerated components.
A. 96-well spot maps
B. 384-well spot maps
Figure 2. Spot Maps 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 and in the data files.
Linkers
Each Linker has a biotin-binding domain that couples to the biotinylated capture antibody, 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.
Record which antibody is coupled to each Linker when performing the coupling step (as described in the Reagent Preparation section).
U-PLEX Antibody Sets
You will receive U-PLEX Antibody Sets containing the biotinylated capture antibody and the SULFO-TAG™ conjugated detection antibody (Table 2).
| A | B | C | D | E | F | G | |
| Name | Storage | Size | Quantity Supplied | Description | |||
| 1 Plate | 5 Plates | 25 Plates | Set containing biotinylated capture antibody and SULFO-TAG conjugated detection antibody | ||||
| U-PLEX Mouse Analyte-Specific Antibody Set | 2–8 °C | 1 Plate | 1 | — | — | ||
| 5 Plate | — | 1 | 5 | ||||
Table 2. Contents of U-PLEX Antibody Sets
Dash (—) = not applicable
U-PLEX Calibrators
Calibrators may be either lyophilized or frozen. Individual analyte concentrations are provided in lot-specific certificates of analysis (COA). Depending on the specific assays requested, one or more of the following Calibrators will be provided (Table 3).
| A | B | C | D | |
| Name | Storage | Catalog No. | Analytes | |
| Calibrator 5 | 2–8 °C | C0065-2 | EPO, GM-CSF, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, KC/GRO, TNF-a, VEGF-A | |
| Calibrator 7 | 2–8 °C | C0073-2 | IL-16, IL-17A, IL-17C, IL-17E/IL-25, IL-21, IL-22, IL-23 | |
| Calibrator 8 | 2–8 °C | C0074-2 | IL-15, IL-17F, IL-31, IL-33, IL-27p28/IL-30 | |
| Calibrator 12 | 2–8 °C | C0092-2 | IL-9, IL-17A/F, IP-10, MCP-1, MIP-1a, MIP-1b, MIP-2, MIP-3a | |
| Calibrator 16 | ≤–70 °C | C0295-2 | 6CKine/CCL21, BAFF, BCA-1/BLC, CD40, IFN-β, MCP-5/CCL12, MDC | |
| Calibrator 17 | 2–8 °C | C0296-2 | Eotaxin, MMP-9 (total), NGAL/LCN2, RANTES, SDF-1α, TARC, TNF-RI | |
| Calibrator 18 | 2–8 °C | C0292-2 | BDNF, C-Peptide, FGF-21, Ghrelin, GLP-1 (inactive), GLP-1 (total), PYY (total) | |
| Calibrator 19 | 2–8 °C | C0293-2 | Glucagon, Insulin, Leptin | |
| Ghrelin (active) | 2–8 °C | C016K-2 | Ghrelin (active) | |
| GLP-1 (active) | 2–8 °C | C016L-2 | GLP-1 (active) | |
| Mouse IFN-α Calibrator | 2–8 °C | C02W1-2 | IFN-α |
Table 3. Analytes included in the Calibrators available for U-PLEX Metabolic Group 1 (mouse)
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 (1,000–1,500 for 384-well plates).
Wash Buffer (20x)MESO SCALE DIAGNOSTICS, LLC.Catalog #R61AA-1 for plate washing. The standard protocol uses a minimum of 415 mL of 1X Wash Buffer for a 384-well plate and 130 mL for a 96-well plate. Automated plate washers may need overage added to these volumes.
- Adhesive plate seals.
- Deionized water.
- A dipeptidyl peptidase IV (DPP-IV) inhibitor (such as diprotin A) is recommended for certain assays.
MSD Blocker D-RMESO SCALE DIAGNOSTICS, LLC.Catalog #R93BR is recommended for certain assays.
Halt™ Protease Inhibitor Cocktail, EDTA-Free (100X)Thermo FisherCatalog #87785 . Recommended for Ghrelin (active) assay. Use as recommended by the manufacturer.
- Vortex mixer.
Note
If including Open Spots, you will also need:
- MSD GOLD SULFO-TAG NHS-Ester (catalog no. R91AO-1) for conjugating detection reagents or SULFO-TAG conjugated antispecies antibodies for use as reporters with unconjugated detection antibodies.
- Sulfo-NHS-LC-Biotin for biotinylating the capture reagents (e.g., EZ-Link Sulfo-NHS-LC-Biotin, Thermo Fisher Scientific, catalog no. 21327, or equivalent).
MSD GOLD SULFO-TAG NHS-EsterMESO SCALE DIAGNOSTICS, LLC.Catalog #R91AO-1
- Zeba Desalting Columns (Thermo Fisher Scientific, catalog numbers 87766-87773).
Zeba™ Spin Desalting Columns, 40K MWCO, 0.5 mLThermo FisherCatalog #87766 Zeba™ Spin Desalting Columns, 40K MWCO, 0.5 mLThermo FisherCatalog #87767 Zeba™ Spin Desalting Columns, 40K MWCO, 2 mLThermo FisherCatalog #87768
Zeba™ Spin Desalting Columns, 40K MWCO, 2 mLThermo FisherCatalog #87769
Zeba™ Spin Desalting Columns, 40K MWCO, 5 mLThermo FisherCatalog #87770
Zeba™ Spin Desalting Columns, 40K MWCO, 5 mLThermo FisherCatalog #87771
Zeba™ Spin Desalting Columns, 40K MWCO, 10 mLThermo FisherCatalog #87772
Zeba™ Spin Desalting Columns, 40K MWCO, 10 mLThermo FisherCatalog #87773
- Coating diluent such as 0.5% bovine serum albumin in PBS, or MSD Diluent 100 for diluting the capture antibody.
Instrument Compatibility
MSD offers U-PLEX Assays designed for use on specific instrument platforms (Table 4).
| A | B | C | |
| Instrument | Assays on U-PLEX 96-well SECTOR™ Plate | Assays on U-PLEX 384-well SECTOR Plate | |
| MESO® QuickPlex SQ 120 | Y | — | |
| MESO QuickPlex® SQ 120MM | Y | — | |
| MESO SECTOR® S 600 | Y | Y | |
| MESO SECTOR S 600MM | Y | Y |
Table 4. Instrument compatibility
Dash (—) = not applicable
Prepare Calibrator Standards
For Lyophilized Calibrators
Bring the Calibrators to room temperature. Reconstitute by adding 250 µL of Metabolic Assay Working Solution to the vial. This will result in a 10X concentrated stock of each Calibrator. Invert the reconstituted Calibrator at least 3 times. Do not vortex. Let the reconstituted solution equilibrate at room temperature for 15–30 minutes and then vortex briefly. The Calibrator is now ready for use.
For Liquid Calibrators
Thaw the stock Calibrator(s) and gently mix. Keep on ice. Once thawed, the Calibrator is ready to use.
Note
We recommend that reconstituted or thawed Calibrators be used immediately. If storage is necessary, divide Calibrators into suitably sized aliquots (60 µL aliquots are recommended) and store immediately at ≤–70 °C.
Prepare Calibrator Standard 1 (top of the curve) in a polypropylene tube by mixing and diluting the reconstituted or thawed Calibrator as indicated in Table 6. Mix by vortexing.
| A | B | C | D | |
| No. of Calibrators Provided | Volume of Reconstituted Calibrator (µL) | Metabolic Assay Working Solution (µL) | Total volume (µL) | |
| 2 | 25 each | 200 | 250 | |
| 3 | 25 each | 175 | 250 | |
| 4 | 25 each | 150 | 250 | |
| N | 25 each | 250 - (25 x N) | 250 |
Table 6. Combining Calibrators to generate the Calibrator Standard 1 (top of the curve) level
Prepare the subsequent 6 dilutions for the curve (4-fold serial dilutions) in Metabolic Assay Working Solution (see Figure 4; Table 7). Use Metabolic Assay Working Solution for the Calibrator Standard 8 (zero Calibrator/blank). Mix by vortexing the tubes between each serial dilution.
| A | B | C | D | E | F | |
| Calibrator Standard No. | Tube No. | Source of Calibrator | Volume of Reconstituted Calibrator (µL) | Metabolic Assay Working Solution (µL) | Total volume (µL) | |
| 1 | 1 | Calibrator Standard 1 (top of curve) | See Table 6 | |||
| 2 | 2 | From tube 1 | 75 | 225 | 300 | |
| 3 | 3 | From tube 2 | 75 | 225 | 300 | |
| 4 | 4 | From tube 3 | 75 | 225 | 300 | |
| 5 | 5 | From tube 4 | 75 | 225 | 300 | |
| 6 | 6 | From tube 5 | 75 | 225 | 300 | |
| 7 | 7 | From tube 6 | 75 | 225 | 300 | |
| 8 (zero Calibrator) | 8 | — | 0 | 300 | 300 |
Table 7. Serial dilution to generate the standard curve
Dash (—) = not applicable
Figure 4. Dilution schema for preparation of Calibrator Standards for U-PLEX Metabolic Group 1 (mouse) Assays.
Sample Collection and Handling
Below are general guidelines for sample collection, storage, and handling for metabolic markers. We strongly suggest following these procedures if working with the active forms of protein analytes. If other methods are used, evaluate sample stability under the selected method as needed.
The assay requires 50 µL/well of the sample (25 μL for 384-well plates). Based on the number of replicates desired, prepare an adequate volume of the sample.
Sample Collection (preferred method): Samples should be collected using the BD P800 Collection and Preservation System, which contains a DPP-IV and other protease inhibitor cocktails (Product Number 366420 or 366421). The alternative collection method described below with K2EDTA tubes can also be used.
Non-P800 collection method: Collect blood in BD Vacutainer K2EDTA Tubes (Product Number 367841 or 366643). Immediately add a DPP-IV inhibitor (0.1 mM final concentration, not provided) and aprotinin (1,000 KIU/mL final concentration) and mix to avoid cleavage/degradation of metabolic peptides.
For BD tubes, process as follows:
In a swing-out rotor centrifuge, spin the blood collection tubes as follows;
• For 2 mL tubes —10 minutes at 1,000 × g (2–8 °C).
• For 8.5 and 10 mL tubes—20 minutes at 1,300 × g (2–8 °C).
Use the plasma immediately or the samples can be stored at 2–8 °C if used within 3 hours. For future use, aliquot the plasma and freeze in suitably sized aliquots at ≤–70 °C.
For samples other than serum and plasma, add a DPP-IV inhibitor (0.1 mM final concentration, not provided) and aprotinin (1,000 KIU/mL final concentration) and use immediately or freeze at –70 °C.
Samples with hemolysis or significant lipemia may hinder accurate measurements.
Repeated freezing and thawing of samples is not recommended. After thawing, centrifuge samples at 2,000 × g for 3 minutes to remove particulates before use in the assay. If the samples are clear and no particulates are visible, you may not need to centrifuge. Hold on wet ice or 2–8 °C until processed and used in the assay.
Dilute Samples
Depending on the sample set under investigation, dilution may be necessary. At least a two-fold dilution of serum and plasma is recommended to reduce matrix effects. Metabolic Assay Working Solution should be used for sample dilution. The dilution factor for the given sample type may need to be optimized.
Note
For BAFF, the concentrations in normal serum and EDTA plasma may exceed the standard working range of the assays. Refer to the product-specific datasheets for additional information.
Wash Buffer
Prepare a 1X working solution by diluting the 20X stock with deionized water.
Read Buffer
MSD provides MSD GOLD Read Buffer B ready to use. Do not dilute.
Safety warnings
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 applicable safety data sheet(s) (SDS), which can be obtained from MSD Customer Service or at www.mesoscale.com.
