Protocol of Anti-drug Antibody (ADA) Bridging ELISA

This method provides a general procedure for generating an anti-drug antibody (ADA) bridging ELISA standard curve with anti-bevacizumab antibody, using bevacizumab antibody for capture and detection. In our protocol, Bevacizumab is only illustrated as an example and could be substituted by Adalimumab, Cetuximab, Denosumab, Eculizumab, Trastuzumab, Vedolizumab and so forth.

Notes:

1. This method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets).
2. This protocol will also need to be adjusted for use with different detection methods and immunoassay technology platforms if required.

Reagents

• PBS: 136 mM NaCl

  2.68 mM KCl
  8.1 mM Na₂HPO₄
  1.46 mM KH₂PO₄

• PBST: PBS with 0.05% Tween-20
• BSA
• Human Serum
• Fluorogenic Peroxidase Substrate

Materials

• 384-well microtiter plate, black, square flat-bottom wells.
• Qualified 96-well plates can be used instead of 384-well plates. For the 96-well format, use 100 μl (instead of 20 μl) of antigen, antibodies, or substrate and 300 μl for the blocking step.
• Fluorescence plate reader.

Method

1. Prepare the unconjugated bevacizumab as capture antibody at 1 µg/ml in PBS. Coat the required number of wells of a 384-well microtiter plate with 20 µl per well of the prepared capture antibody. Incubate overnight at 4°C
2. Wash the microtiter plate five times with PBST.
3. Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hour at room temperature (RT).
4. Wash the microtiter plate five times with PBST.
5. For the standard curve, prepare a dilution series of the anti-bevacizumab antibody in 10% human serum in PBST in triplicate. Final concentration of anti-bevacizumab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. A zero anti-bevacizumab concentration as the background value is required.
6. Add 20 μl of each of the diluted antibody standards to the wells designated for the standard curve (in triplicate for each standard recommended). Add 20 μl of each test sample to the other wells (in triplicate for each sample recommended). Incubate for 1 hour at RT.
7. Wash the microtiter plate five times with PBST.
8. To each well, add 20 µl pre-prepared HRP conjugated bevacizumab diluted to 2 µg/ml in dilution buffer and incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. Add 20 μl substrate to each well and measure the fluorescence after 30 minutes.
11. Data analysis.

Note: The concentrations involved in this scheme may need to be adjusted according to the actual situation.

Click here for the Troubleshooting of Anti-drug Antibody (ADA) Bridging ELISA.