Protocol: PK Bridging ELISA For Use With Anti-Trastuzumab Antibodies HCA169, HCA270

Reagents

BSA

HISPEC immunoassay diluent (BUF049)

Human Serum (Sigma-Aldrich, H4522)

Mouse anti-human IgG4:HRP (MCA2098P)

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 the anti-trastuzumab capture antibody HCA169 (AbD18141) at 1 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 antibody, and 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 concentration of trastuzumab should cover the range from 0.125 ng/ml to 8,000 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 detection antibody HCA270 (AbD18018_hIgG4_Pro) at 2 μg/ml in PBST. Incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. To each well, add 20 μl HRP conjugated mouse anti-human IgG4 antibody, MCA2098P diluted 1:5,000 in HISPEC buffer and 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.

V1.12.2016