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

Reagents

BSA

HISPEC Assay Diluent  (BUF049)

Human Serum (Sigma-Aldrich, H4522)

LYNX Rapid HRP Antibody Conjugation Kit® (#LNK001P-LNK006P)

PBS

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

PBST

PBS with 0.05% Tween 20

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, 437111). 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 detection antibody: conjugate pembrolizumab antibody using a LYNX Rapid HRP Antibody Conjugation Kit.
2. Prepare the unconjugated pembrolizumab 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, 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 an Anti-Pembrolizumab Antibody HCA297 (AbD30685_hIgG1) or HCA298 (AbD30689_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-pembrolizumab antibody should cover the range
   from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-pembrolizumab antibody concentration as the background value.
7. Add 20 μl of anti-pembrolizumab 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 pembrolizumab diluted to 2 μg/ml in HISPEC Assay Diluent 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.