Protocol: PK Antigen Capture ELISA for Use with Anti-Atezolizumab Antibodies
Pharmacokinetic (PK) Antigen Capture ELISA: For use with Anti-Atezolizumab Monoclonal Antibody catalog TZA013
This method provides a procedure for carrying out a PK ELISA antigen capture format with Anti-Atezolizumab Antibody, TZA013 (detection antibody), and using atezolizumab for the standard curve. Anti-atezolizumab drug/target complex antibody recognizes atezolizumab 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.
View all of our anti-atezolizumab antibodiesReagents
- BSA (Sigma-Aldrich, A7906-100)
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 (Merck Millipore, 817072)
- QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
Recombinant Human PD-L1 Protein (Sino Biologicals, 10084-H08H)
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
- 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.
- Wash the microtiter five times (5x) with PBST.
- Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
- Wash the microtiter plate 5x with PBST.
- For the standard curve, prepare a dilution series of atezolizumab in 10% human serum in PBST in triplicate. Final concentration of atezolizumab should cover the range from 0.01 ng/ml to 10,000 ng/ml. Include a zero atezolizumab concentration as the background value.
- Add 20 μl of atezolizumab 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.
- Wash the microtiter plate 5x with PBST.
- To each well, add 20 μl detection Anti-Atezolizumab Antibody, TZA013 (AbD40640ad) at 2 μg/ml in PBST. Incubate for 1 hr at RT.
- Wash the microtiter plate 5x with PBST.
- To each well, add 20 μl Anti-FLAG M2-Peroxidase HRP Antibody (Sigma-Aldrich, A8592) at a 1:20,000 dilution in HISPEC Assay Diluent (BUF049). Incubate for 1 hour at RT.
- Wash the microtiter plate 10x with PBST.
- Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169) to each well and measure the fluorescence after 30 min.
