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

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

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

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

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Pharmacokinetic (PK) Antigen Capture Format ELISA: For use with Anti-Pertuzumab Monoclonal Antibody catalog TZA035P

This method provides a procedure for carrying out a PK ELISA antigen capture format with HRP-conjugated Anti-Pertuzumab Antibody TZA035P (detection antibody), using pertuzumab for the standard curve. Anti-pertuzumab drug/target complex antibody recognizes pertuzumab only when bound to its target human HER2. It does not recognize the free drug or unbound human HER2. 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)
​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)

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 HER2 (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 pertuzumab in 10% human serum in PBST in triplicate. Final concentration of pertuzumab should cover the range from 0.001 ng/ml to 1,000 ng/ml. Include a zero pertuzumab concentration as the background value.
  6. Add 20 µl of pertuzumab 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 HRP-conjugated Anti-Pertuzumab Antibody, TZA035P (AbD35572pap) at 0.5 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.