Protocol: PK Antigen Capture ELISA for Use with Anti-ipilimumab Antibody TZA001

Protocol: PK Antigen Capture ELISA for Use with Anti-ipilimumab Antibody TZA001

Pharmacokinetic (PK) Antigen Capture ELISA: for Use with Anti-Ipilimumab Monoclonal Antibody TZA001

Protocol: PK Bridging ELISA for Use with Anti-ipilimumab Antibody TZA001P

Protocol: PK Antigen Capture ELISA for Use with Anti-Ipilimumab Antibody

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This method provides a procedure for carrying out a PK ELISA antigen capture format with Anti-Ipilimumab Antibody, TZA001 (detection antibody), and using ipilimumab for the standard curve. Anti-ipilimumab drug/target complex antibody recognizes ipilimumab only when bound to its target, human CTLA-4. It does not recognize the free drug or unbound human CTLA-4. 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 (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 CTLA-4 Protein (Sino Biological, 1159-H03H)
Anti-FLAG-Tag: 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 human CTLA-4 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 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 ipilimumab in 10% human serum in PBST in triplicate. Final concentration of ipilimumab should cover the range from 0.001 ng/ml to 4,000 ng/ml. Include a zero ipilmumab concentration as the background value.
  6. Add 20 μl of ipilimumab dilution per well (in triplicate for each standard is recommended). Add 20 µl of each test sample to the other wells (in triplicate for each sample is recommended). Incubate for 1 hr at RT.
  7. Wash the microtiter plate 5x with PBST.
  8. To each well, add 20 µl detection Anti-Ipilimumab Antibody, TZA001 (AbD34294) 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-TAG:HRP Antibody, (Sigma-Aldrich, A8592) 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 (Thermo Fisher Scientific, 15169) to each well and measure the fluorescence after 30 min