Protocol: ADA Bridging ELISA For Use With Anti-Cetuximab Antibody

Reagents

BSA

HISPEC immunoassay diluent (Bio-Rad, BUF049)

Human Serum (Sigma-Aldrich, H4522)

Lynx Rapid HRP Antibody Conjugation Kit® (Bio-Rad, 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, MaxiSorp™ PS (Thermo Fisher Scientific, 460518)

Fluorescence plate reader

96-well plates can be used instead of 384-well plates, e.g. black, flat-bottom MaxiSorp PS (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 cetuximab antibody using a Lynx Rapid HRP Antibody Conjugation Kit.
2. Prepare the unconjugated cetuximab 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 cetuximab, 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 the anti-cetuximab antibody HCA221 (AbD19830_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-cetuximab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-cetuximab concentration as the background value.
7. Add 20 μl of anti-cetuximab 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 cetuximab diluted to 2 µg/ml in HISPEC buffer and incubate for 1 hour at RT.
10. Wash the microtiter plate ten times with PBST.
11. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V4.3.2016