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

Reagents

BSA
HISPEC Assay Diluent (Bio-Rad, BUF049)
Mouse anti-Penta Histidine Tag:HRP (Bio-Rad, MCA5995P)
Human Serum (Sigma-Aldrich, H4522)
LYNX Rapid HRP Antibody Conjugation Kit® (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