Protocol: ADA Bridging ELISA for Use with Anti-Golimumab Antibodies
Protocol: ADA Bridging ELISA for Use with Anti-Golimumab Antibodies
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Anti-Drug Antibody (ADA) Bridging ELISA: for use with Anti-Golimumab Monoclonal Antibody HCA287
This method provides a procedure for generating an ADA ELISA standard curve with Anti-Golimumab Antibody, HCA287, using golimumab 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 Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)
LYNX Rapid HRP Antibody Conjugation Kit® (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, 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
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Prepare golimumab for detection: conjugate golimumab using a LYNX Rapid HRP Antibody Conjugation Kit.
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Prepare unconjugated golimumab 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 golimumab solution, and incubate overnight at 4°C.
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Wash the microtiter plate five times (5x) with PBST.
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Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
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Wash the microtiter plate 5x with PBST.
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For the standard curve, prepare a dilution series of the Anti-Golimumab Antibody, HCA287 (AbD25418_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of Anti-Golimumab Antibody should cover the range from 1 ng/ml to 32,000 ng/ml. Include a zero Anti-Golimumab Antibody concentration as the background value.
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Add 20 µl of Anti-Golimumab Antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hr at RT.
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Wash the microtiter plate 5x with PBST.
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To each well, add 20 µl HRP conjugated golimumab diluted to 2 µg/ml in HISPEC Assay Diluent and incubate for 1 hr at RT.
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Wash the microtiter plate 10x with PBST.
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Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.