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

Pharmacokinetic (PK) bridging ELISA protocol detects total drug (free, partially and fully bound): For use with Anti-Infliximab Antibody products HCA214 and HCA216P

This method provides a procedure for carrying out a PK ELISA with Anti-Infliximab Antibodies, catalog numbers HCA214 (capture antibody) and HCA216P (detection antibody), and using infliximab for the standard curve. These two antibodies do not inhibit the binding of infliximab to TNFα, therefore this assay can 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 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, 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

Prepare the capture Anti-Infliximab Antibody HCA214 (AbD19376) 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 capture antibody, and incubate overnight at 4°C.
Wash the microtiter plate five times (5x) with PBST.
Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
Wash the microtiter plate 5x with PBST.
For the standard curve, prepare a dilution series of infliximab in 10% human serum in PBST in triplicate. Final concentration of infliximab should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero infliximab concentration as the background value.
Add 20 μl of infliximab dilution per well (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 hr at RT.
Wash the microtiter plate 5x with PBST.
To each well, add 20 µl HRP conjugated detection Anti-Infliximab Antibody, HCA216P (AbD19376_hIgG1) at 2 µg/ml in HISPEC Assay Diluent. Incubate for 1 hr at RT.
Wash the microtiter plate 10x with PBST.
Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.