Protocol: ADA Bridging ELISA for use with Anti-Etanercept Antibody

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, e.g. 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) e.g. 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 etanercept using a LYNX Rapid HRP Antibody Conjugation Kit.
2. Prepare unconjugated etanercept 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 etanercept solution, and incubate overnight at 4°C.
3. Wash the microtiter plate five times with PBST.
4. 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).
5. Wash the microtiter plate five times with PBST.
6. For the standard curve, prepare a dilution series of Anti-Etanercept Antibody, HCA277 (clone AbD25940_hIgG4Pro) in 10% human serum in PBST in triplicate. Final concentration of anti-etanercept antibody should cover the range from 1 ng/ml to 32,000 ng/ml. Include a zero anti-etanercept antibody concentration as the background value.
7. Add 20 µl of anti-etanercept antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
8. Wash the microtiter plate five times with PBST.
9. To each well, add 20 µl HRP conjugated etanercept diluted to 2 µg/ml in HISPEC Assay Diluent and incubate for 1 hour at RT.
10. Wash the microtiter plate ten times with PBST.
11. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 minutes.