Protocol: PK Bridging ELISA For Use With Anti-Infliximab Antibody

Protocol: PK Bridging ELISA for Use with Anti-Nivolumab Antibodies

Protocol: PK Bridging ELISA for Use with Anti-Nivolumab Antibodies

Protocol: PK Bridging ELISA for Use with Anti-Nivolumab Antibodies

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Pharmacokinetic (PK) Bridging ELISA: for Use with Anti-Nivolumab Monoclonal Antibodies HCA299 and HCA301P

This method provides a procedure for carrying out a PK ELISA with Anti-Nivolumab Antibodies, catalog number HCA299 (capture antibody) and HCA301P (detection antibody), and using nivolumab for the standard curve. 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.


HISPEC Assay Diluent  (BUF049)
Human Serum (Sigma-Aldrich, H4522)
136 mM NaCl
2.68 mM KCl
8.1 mM Na2HPO4
1.46 mM KH2PO4
PBS with 0.05% Tween 20
QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)


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.


  1. Prepare the capture Anti-Nivolumab Antibody HCA299 (AbD30255) 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 capture antibody, and 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 nivolumab in 10% human serum in PBST in triplicate. Final concentration of nivolumab should cover the range from 0.125 ng/ml to 8,000 ng/ml. Include a zero nivolumab concentration as the background value.
  6. Add 20 μl of nivolumab dilution per well (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 HRP conjugated detection Anti-Nivolumab Antibody, HCA301P (AbD30258_hIgG1) at 2 μg/ml in HISPEC Assay Diluent. Incubate for 1 hr at RT.
  9. Wash the microtiter plate 10x with PBST.
  10. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.