Protocol: PK ELISA Antigen Capture Format 

For Use With Anti-Trastuzumab Antibody

Protocol: PK ELISA Antigen Capture Format For Use With Anti-Trastuzumab Antibody

Protocol: PK ELISA Antigen Capture Format For Use With Anti-Trastuzumab Antibody

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Pharmacokinetic (PK) ELISA Antigen Capture Format: For use with anti-trastuzumab monoclonal antibody product HCA263

This method provides a procedure for carrying out a PK ELISA Antigen Capture Format with anti-trastuzumab antibody, product code HCA263 (detection antibody), and using trastuzumab monoclonal antibody for the standard curve. Anti-trastuzumab drug/target complex antibody recognizes trastuzumab only when bound to its target human ErbB2. It does not recognize the free drug or unbound human ErbB2. 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 immunoassay diluent (Bio-Rad, BUF049)
Mouse anti-Penta Histidine Tag:HRP (Bio-Rad, MCA5995P)
Human Serum (Sigma-Aldrich, H4522)
Lynx Rapid HRP Antibody Conjugation Kit® (Bio-Rad, LNK001P-LNK006P)
For best results when conjugating with Lynx Rapid HRP Antibody Conjugation Kit, avoid using antibody with thiomersal as preservative. Contact us to discuss thiomersal-free options. 
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, MaxiSorp™ PS (Thermo Fisher Scientific, 460518)
Fluorescence plate reader
96-well plates can be used instead of 384-well plates, e.g. black, flat-bottom MaxiSorp PS (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 ErbB2 (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. Incubate overnight at 4°C.
  2. Wash the microtiter plate five times with PBST.
  3. Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hour at room temperature (RT).
  4. Wash the microtiter plate five times with PBST.
  5. For the standard curve, prepare a dilution series of trastuzumab in 10% human serum in PBST in triplicate. Final concentrations of trastuzumab should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero trastuzumab concentration as the background value.
  6. Add 20 μl of each of the diluted standards to the wells designated for the standard curve (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 hour at RT.
  7. Wash the microtiter plate five times with PBST.
  8. To each well, add 20 μl human anti-trastuzumab antibody HCA263 (AbD25279) at a concentration of 2 μg/ml in PBST
    buffer. Incubate for 1 hour at RT.
  9. Wash the microtiter plate ten times with PBST.
  10. To each well, add 20 μl anti-Penta Histidine Tag:HRP (MCA5995P) at a 1:2000 dilution in HISPEC buffer. Incubate for 1 hour at RT.
  11. Wash the microtiter plate ten times with PBST.
  12. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V1.11.2015


Lynx Rapid Conjugation Kit® is a registered trademark of Bio-Rad AbD Serotec Ltd.
MaxiSorp™ and QuantaBlu™ are trademarks of Thermo Fisher Scientific.
Tween® is a registered trademark of ICI Americas Inc.