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

Reagents

BSA (Sigma-Aldrich, A7906)
​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 (Merck Millipore, 817072)

QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
Atezolizumab

 

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 the detection antibody: conjugate sarilumab antibody using a LYNX Rapid HRP Antibody Conjugation Kit (LNK001P-LNK006P).
2. Prepare the unconjugated sarilumab 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 sarilumab, 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-Sarilumab Antibody HCA410 (AbD49514ia), HCA411 (AbD49516ia), or HCA412 (AbD49520ia) in 10% human serum in PBST in triplicate. Final concentration of anti-sarilumab antibody should cover the range from 0.1 ng/ml to 100,000 ng/ml. Include a zero anti-sarilumab antibody concentration as the background value.
7. Add 20 µl of anti-sarilumab 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 sarilumab 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.