Protocol: ADA Bridging ELISA For Use With Anti-Trastuzumab Antibody
Protocol: ADA Bridging ELISA For Use With Anti-Trastuzumab Antibody
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Anti-Drug Antibody (ADA) Bridging ELISA: For use with anti-trastuzumab monoclonal antibody product HCA177
This method provides a procedure for generating an ADA ELISA standard curve with anti-trastuzumab antibody, product code HCA177, using trastuzumab 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
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Prepare detection antibody: conjugate trastuzumab antibody using a Lynx Rapid HRP Antibody Conjugation Kit.
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Prepare the unconjugated trastuzumab capture antibody at 0.5 µg/ml in PBS. Coat the required number of wells of a 384-well microtiter plate with 20 µl per well of the prepared trastuzumab. Incubate overnight at 4°C.
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Wash the microtiter plate five times 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 hour at room temperature (RT).
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Wash the microtiter plate five times with PBST.
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For the standard curve, prepare a dilution series of the anti-trastuzumab antibody HCA177 (AbD18018_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-trastuzumab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-trastuzumab concentration as the background value.
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Add 20 µl of anti-trastuzumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
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Wash the microtiter plate five times with PBST.
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To each well add 20 µl HRP conjugated trastuzumab diluted to 2 µg/ml in HISPEC buffer and incubate for 1 hour at RT.
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Wash the microtiter plate ten times with PBST.
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Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.
V3.3.2016
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