Protocol: PK Bridging ELISA for use with Anti-Etanercept Antibodies

Reagents

BSA
​HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)

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 the capture Anti-Etanercept Antibody HCA276 (AbD25939) at 5 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.
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 etanercept in 10% human serum in PBST in triplicate. Final concentration of etanercept should cover the range from 0.01 ng/ml to 1,000 ng/ml. Include a zero etanercept concentration as the background value.
6. Add 20 μl of each of the diluted 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 HRP conjugated detection Anti-Etanercept Antibody HCA277P (AbD25940_hIgG4Pro) diluted to 2 µg/ml in HISPEC Assay Diluent and incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 minutes.