2. Principle of the Assay

Biotinylated capture antibodies are coupled to one of ten unique 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 MSD’s electrochemiluminescent (ECL) labels (MSD GOLD™ SULFO-TAG) bind to the analytes to complete the sandwich immunoassay (shown below). Once the sandwich immunoassay is complete, the U-PLEX plate is loaded into the 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.

Electrochemiluminescence Technology

• Minimal non-specific background and strong responses to analyte yield high signal-to-background ratios.
• The stimulation mechanism (electricity) is decoupled from the response (light signal), minimizing matrix interference. 
• Only labels bound near the electrode surface are excited, enabling non-washed assays.
• Labels are stable, non-radioactive, and directly conjugated to biological molecules.
• Emission at ~620 nm eliminates problems with color quenching.
• Multiple rounds of label excitation and emission enhance light levels and improve sensitivity.
• Carbon electrode surface has 10X greater binding capacity than polystyrene wells.
• Surface coatings can be customized.

3. U-PLEX Assay Protocol

STEP 1:  Create Individual U-PLEX Coupled Antibody Solutions

• Add 200 µL of each biotinylated antibody to 300 µL of the assigned linker. Incubate at room temperature (RT) for 30 minutes. 
• Add 200 µL of Stop Solution. Incubate at RT for 30 minutes.

STEP 2:  Prepare the Multiplex Coating Solution

• Combine 600 µL of each U-PLEX coupled antibody solution. Up to 10 U-PLEX coupled antibodies can be pooled. Bring the solution up to 6 mL with Stop Solution.

STEP 3:  Coat the U-PLEX Plate

• Add 50 µL of multiplex coating solution to each well. Seal the plate and incubate at RT while shaking for 1 hour.  Wash the plate 3 times with 150 µL/well Wash Buffer.

STEP 4:  Add Sample or Calibrator Standard

• Add 25 µL of appropriate diluent to each well. Add 25 µL of prepared sample or calibrator standard to each well. Seal the plate and incubate at RT with shaking for 1 hour.

STEP 5:  Wash and Add Detection Antibody Solution

• Wash plate 3 times with 150 µL/well of Wash Buffer. 
• Add 150 µL of detection antibody solution to each well. Seal the plate and incubate at RT with shaking for 1 hour.

STEP 6:  Wash and Read

• Wash plate 3 times with 150 µL/well of Wash Buffer. 
• Add 150 µL 2X Read Buffer T to each well.  Analyze plate on the MSD instrument.

4. Screening for Viable Antibody Pairs Using the U-PLEX Platform

Unbiased screening of 59 antigen-specific antibody pairs on the U-PLEX platform. Pair-wise combinations of all antibodies labeled with biotin or MSD GOLD SULFO-TAG were evaluated for their ability to produce robust signal in the presence of calibrator and low signal in the absence of calibrator (diluent only). Shown is a heat map of selected signal data from this study arranged to allow visualization of antibody pairs that generated the highest signals. Green represents pairs with the highest signals, yellow represents pairs with signal in the 50th percentile and red represents pairs with low or no signal. 38 antibody pairs were selected from this screen based on high signal and low background (data not shown) for continued screening.

5. Secondary Screening of Selected Antibody Pairs on the U-PLEX Platform

6. Sample Testing

Sample testing on the U-PLEX platform. A) Concentrations of analytes detected by each proposed antibody pair. B) Relevant standard curve values for each antibody pair, including LLOD (selected pairs highlighted in yellow).  
The nine selected antibody pairs were evaluated for their ability to detect analyte in 12 different stimulated samples. Stimulated samples were used because the analyte is not present in normal matrices. Full standard curves and stimulated samples were run in duplicate. All pairs were able to detect analyte in samples 2, 5 and 7. Three pairs (highlighted by red arrows) were selected based on best reactivity to samples and overall assay sensitivity.

7. Matrix Tolerance

Three antibody pairs from the original nine were selected to evaluate matrix tolerance on the U-PLEX platform.

A) To assess dilution linearity, normal human serum (n=3) and EDTA plasma samples (n=3) and diluent (n=3) were spiked with recombinant calibrator and diluted 2-, 4-, 8-. and 16-fold before testing. The average percent recovery is based on samples that measured within the quantitative range of each assay. All three antibody pairs were found to recover within 20% of the targeted range, suggesting that the samples dilute linearly 2- to 16-fold.
B) Spike recovery was evaluated by spiking normal human serum (n=3) and EDTA plasma (n=3) from a commercial source or diluent with calibrators at three different levels (high, mid, and low).  All three antibody pairs were found to recover within 20% of the targeted range. Overall, the data suggested that the three selected antibody pairs were not sensitive to matrix effects. Of these three viable antibody pairs, Capture 1/Detect 2 and Capture 2/Detect 2 were selected for optimization.

8. Assay Range

Characterization of assay range for one of selected antibody pairs (Capture 1/Detect 2). A twelve point standard curve was prepared with an initial top of curve concentration of 40,000 pg/mL. A) Standard curve for proposed primary antibody pair. Pair shows 3- to 4-logs of dynamic range.  B) Signals and %CVs of each point in the proposed primary assay standard curve.

9. Detection Antibody Titration

Detection antibody titration optimization on the U-PLEX platform. Detection antibody titration was performed with antibody pair (Capture 1/Detect 2) selected from screening. Resultant data was used to select an optimal detection antibody concentration, which in this case was 1 µg/mL.

10. Assay Specificity

Assay specificity testing on the U-PLEX platform. The selected assay pair (Capture 1/Detect 2) was multiplexed with six additional assays to evaluate calibrator/antibody specificity.  In this study, the captured antibody for each assay was linked to individual spots in wells on the U-PLEX plate and tested with individual calibrators and a blended detection antibody solution. The data demonstrated that non-specific binding (non-specific binding signal/specific signal X 100) was less than 0.5% for all of the assays. Overall, the data indicated that the new selected antibody pair can be successfully multiplexed with six additional assays to form a potential U-PLEX panel.

11. Conclusion

We demonstrated the utility of the MSD U-PLEX platform for the rapid screening and selection of viable antibody pairs from over 3,000 potential antibody combinations. The platform was subsequently used to select and optimize multiple antibody pairs based on parameters such as dynamic range, sensitivity, sample recognition, matrix tolerance, and non-specific binding. These studies demonstrated that the U-PLEX platform can be adapted to facilitate both large and small immunoassay development programs.