Protocol: ADA Bridging ELISA for Use with Anti-Avelumab Antibodies

Reagents

BSA (Sigma-Aldrich, A7906)
​HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)
LYNX Rapid HRP Antibody Conjugation Kit® (LNK001P-LNK006P)
For best results when conjugating with LYNX Rapid HRP Antibody Conjugation Kit, avoid using antibody with thiomersal as preservative. Contact us to discuss thiomersal-free options

PBS

136 mM NaCl
2.68 mM KCl
8.1 mM Na₂HPO₄
1.46 mM KH₂PO₄

PBST

PBS with 0.05% Tween 20 (Merck Millipore, 817072)

QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)

 

Materials

384-well microtiter plate, black, square flat-bottom wells, for example, Black 384-Well Immuno Plates (Thermo Fisher Scientific, 460518)

Fluorescence plate reader

96-well plates can be used instead of 384-well plates (black, flat-bottom wells), for example, Black 96-Well Immuno Plates (Thermo Fisher Scientific, 460518). For the 96-well format use 100 µl (instead of 20 µl) of antigen, antibodies, or substrate and 300 µl for the blocking step.

Method

1. Prepare the detection antibody: conjugate avelumab antibody using a LYNX Rapid HRP Antibody Conjugation Kit (LNK001P-LNK006P).
2. Prepare the unconjugated avelumab 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 avelumab, and incubate overnight at 4°C. 
3. Wash the microtiter plate five times (5x) with PBST.
4. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
5. Wash the microtiter plate 5x with PBST.
6. For the standard curve, prepare a dilution series of the Anti-Avelumab Antibody HCA396 (AbD40519ia), HCA397 (AbD40525ia), or HCA398 (AbD40526ia) in 10% human serum in PBST in triplicate. Final concentration of anti-avelumab antibody should cover the range from 1 ng/ml to 15,000 ng/ml. Include a zero anti-avelumab antibody concentration as the background value. 
7. Add 20 µl of anti-avelumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hr at RT. 
8. Wash the microtiter plate 5x with PBST. 
9. To each well add 20 µl HRP conjugated avelumab diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049) and incubate for 1 hr at RT. 
10. Wash the microtiter plate 10x with PBST.
11. Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.