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

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

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

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

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Pharmacokinetic (PK) Antigen Capture ELISA: for Use with Anti-Ranibizumab Monoclonal Antibodies HCA304, HCA306 and HCA307

This method provides a procedure for carrying out a PK antigen capture ELISA with Anti-Ranibizumab Antibodies, catalog numbers HCA304, HCA306 or HCA307, and using ranibizumab for the standard curve. Anti-Ranibizumab/VEGF Antibody HCA304 recognizes ranibizumab only when bound to its target, human VEGF-A. Anti-Ranibizumab Antibodies HCA306 and HCA307 are non-inhibitory and bind the drug alone and the drug in complex with VEGF-A. 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)
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)
Recombinant Human VEGF Protein (PHP293)
Anti-FLAG M2-Peroxidase (HRP) Antibody (Sigma-Aldrich A8592)

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 Recombinant Human VEGF Protein (capture antigen, PHP293) at 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 capture antigen. Incubate overnight at 4°C.
  2. Wash the microtiter plate five times (5x) with PBST.
  3. Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
  4. Wash the microtiter plate 5x with PBST.
  5. For the standard curve, prepare a dilution series of ranibizumab in 10% human serum in PBST in triplicate. Final concentrations of ranibizumab should cover the range from 0.05 ng/ml to 2,000 ng/ml. Include a zero ranibizumab concentration as the background value.
  6. Add 20 μl of each of the diluted standards to the wells designated for the standard curve (in triplicate for each standard recommended). Add 20 μl of each test sample to the other wells (in triplicate for each sample recommended). Incubate for 1 hr at RT.
  7. Wash the microtiter plate 5x with PBST.
  8. To each well, add 20 μl Human Anti-Ranibizumab Antibody HCA304 (AbD29928), HCA306 (AbD29858), or HCA307 (AbD29865) at a concentration of 2 μg/ml in PBST buffer. Incubate for 1 hr at RT.
  9. Wash the microtiter plate 10x with PBST.
  10. To each well, add 20 μl Anti-FLAG M2-Peroxidase (HRP) Antibody at a 1:20,000 dilution in HISPEC Assay Diluent. Incubate for 1 hr at RT.
  11. Wash the microtiter plate 10x with PBST.
  12. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.