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Protocol: PK Bridging ELISA detecting total drug using anti-cetuximab antibody

Protocol: PK Bridging ELISA Detecting Total Drug Using Anti-Cetuximab Antibodies

PK Bridging ELISA Detecting Total Drug Using Anti-Cetuximab Antibody

Pharmacokinetic (PK) Bridging ELISA protocol detects total drug (free, partially and fully bound): For use with anti-cetuximab monoclonal antibody products HCA220 and HCA228P

This method provides a procedure for carrying out a PK ELISA with anti-cetuximab antibodies, product codes HCA220 (capture antibody) and HCA228P (detection antibody), and using cetuximab monoclonal antibody for the standard curve.

HCA228P anti-cetuximab antibody does not inhibit the binding of cetuximab to EGFR and can therefore detect total drug – free, partially bound and fully bound. The method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets). This protocol will need to be adjusted for use with different detection methods and immunoassay technology platforms.

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Reagents

BSA; HISPEC immunoassay diluent (Bio-Rad, 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 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

Prepare the anti-cetuximab capture antibody HCA220 (AbD19834) 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. Incubate overnight at 4°C.
Wash the microtiter plate five times with PBST.
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).
Wash the microtiter plate five times with PBST.
For the standard curve, prepare a dilution series of cetuximab in 10% human serum in PBST in triplicate. Final concentrations of cetuximab should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero cetuximab concentration as the background value.
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.
Wash the microtiter plate five times with PBST.
To each well, add 20 µl HRP conjugated detection antibody HCA228P (AbD19376_hIgG1) at 2 µg/ml in HISPEC buffer. Incubate for 1 hour at RT.
Wash the microtiter plate ten times with PBST.
Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.