Protocol: PK Antigen Capture ELISA for Use with Anti-Ranibizumab Antibodies

Protocol: ADA Bridging ELISA for Use with Anti-Ranibizumab Antibodies

Protocol: ADA Bridging ELISA for Use with Anti-Ranibizumab Antibodies

Protocol: ADA Bridging ELISA for Use with Anti-Ranibizumab Antibodies

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Anti-Drug Antibody (ADA) Bridging ELISA: for Use with Anti-Ranibizumab Antibody HCA305 and Anti-Bevacizumab Antibody HCA185

This method provides a procedure for generating an ADA ELISA standard curve with an Anti-Ranibizumab Antibody, catalog number HCA305 or Anti-Bevacizumab Antibody HCA185 using ranibizumab antibody for capture and detection. Ranibizumab is a monovalent Fab fragment antibody, and was created from the same parent mouse antibody as bevacizumab. Anti-Bevacizumab Antibody HCA185 ​also recognizes ranibizumab. 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 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

  1. Prepare detection antibody: conjugate ranibizumab antibody using a LYNX Rapid HRP Antibody Conjugation Kit.
  2. Prepare the unconjugated ranibizumab 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 ranibizumab, and incubate overnight at 4°C.
  3. Wash the microtiter plate five times (5x) with PBST.
  4. Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
  5. Wash the microtiter plate 5x with PBST.
  6. For the standard curve, prepare a dilution series of the Anti-Ranibizumab Antibody HCA305 (AbD16746_hIgG1) or the Anti-Bevacizumab Antibody HCA185 (AbD17976_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-ranibizumab or anti-bevacizumab antibody should cover the range from 0.5 ng/ml to 10,000 ng/ml. Include a zero anti-ranibizumab or anti-bevacizumab antibody concentration as the background value.
  7. Add 20 μl of anti-ranibizumab or anti-bevacizumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hr at RT.
  8. Wash the microtiter plate 5x with PBST.
  9. To each well add 20 μl HRP conjugated ranibizumab diluted to 2 μg/ml in HISPEC Assay Diluent and incubate for 1 hr at RT.
  10. Wash the microtiter plate 10x with PBST.
  11. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.