Protocol: PK Capture ELISA for Use with Anti-Durvalumab Antibodies

Protocol: PK Capture ELISA for Use with Anti-Durvalumab Antibodies

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

Protocol: Pharmacokinetic (PK) ELISA Antigen Capture Format

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Phamacokinetic (PK) ELISA Antigen Capture Format: for Use with Anti-Durvalumab Antibody TZA002

This method provides a procedure for carrying out a PK ELISA antigen capture format with Anti-Durvalumab Antibody, TZA002 (detection antibody), and using durvalumab for the standard curve. Anti-durvalumab drug/target complex antibody recognizes durvalumab only when bound to its target human programmed cell death ligand 1 (PD-L1). It does not recognize the free drug or unbound human PD-L1. 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)
Phosphate buffered saline (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 human PD-L1 protein (capture antigen) 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, 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 durvalumab in 10% human serum in PBST in triplicate. Final concentration of durvalumab should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero durvalumab concentration as the background value.
  6. Add 20 µl of durvalumab 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 detection Anti-Durvalumab Antibody, TZA002 (AbD41240) at 2 µg/ml in PBST. Incubate for 1 hr at RT.
  9. Wash the microtiter plate 5x 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 hour 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.