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

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

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

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

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Anti-Drug Antibody (ADA) Bridging ELISA: For use with anti-palivizumab monoclonal antibody HCA262

This method provides a procedure for generating an ADA ELISA standard curve with anti-palivizumab antibody, product code HCA262, using palivizumab 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.


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 step

Method

  1. Prepare detection antibody: conjugate palivizumab antibody using a Lynx Rapid HRP Antibody Conjugation Kit.
  2. Prepare the unconjugated palivizumab 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 palivizumab, and incubate overnight at 4°C.
  3. Wash the microtiter plate five times with PBST.
  4. 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).
  5. Wash the microtiter plate five times with PBST.
  6. For the standard curve, prepare a dilution series of the anti-palivizumab antibody HCA262 (AbD23967_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-palivizumab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-palivizumab concentration as the background value.
  7. Add 20 μl of anti-palivizumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
  8. Wash the microtiter plate five times with PBST.
  9. To each well add 20 μl HRP conjugated palivizumab diluted to 2 μg/ml in HISPEC buffer and incubate for 1 hour at RT.
  10. Wash the microtiter plate ten times with PBST.
  11. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V2.3.2016


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