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

Reagents

BSA

HISPEC immunoassay diluent (Bio-Rad, BUF049)

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)

Recombinant human TNFα (Bio-Rad, PHP051)

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 detection antibody: conjugate the antibody HCA274 (AbD25705, monovalent Fab) or HCA245 (AbD20893_hIgG1) using a Lynx Rapid HRP Antibody Conjugation Kit.
2. Prepare the human capture antigen TNFα 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.
3. Wash the microtiter plate five times with PBST.
4. 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).
5. Wash the microtiter plate five times with PBST.
6. For the standard curve, prepare a dilution series of golimumab in 10% human serum in PBST in triplicate. Final concentrations of golimumab should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero golimumab concentration as the background value.
7. 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.
8. Wash the microtiter plate five times with PBST.
9. To each well, add 20 µl pre-prepared HRP conjugated detection antibody at 2 µg/ml in HISPEC buffer. Incubate for 1 hour at RT.
10. Wash the microtiter plate ten times with PBST.
11. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V6.12.2016