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

Anti-Drug Antibody (ADA) Bridging ELISA: For use with anti-natalizumab monoclonal antibody product HCA249

This method provides a procedure for generating an ADA ELISA standard curve with anti-natalizumab antibody, product code HCA249, using natalizumab antibody for capture and detection. 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); 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.

Method

Prepare detection antibody: conjugate natalizumab antibody using a Lynx Rapid HRP Antibody Conjugation Kit.
Prepare the unconjugated natalizumab 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 natalizumab, and 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 the anti-natalizumab antibody HCA249 (AbD21375_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-natalizumab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-natalizumab concentration as the background value.
Add 20 µl of anti-natalizumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
Wash the microtiter plate five times with PBST.
To each well add 20 µl HRP conjugated natalizumab diluted to 2 µg/ml in HISPEC buffer and 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.