Protocol: ADA Bridging ELISA for Use with Anti-Belimumab Antibody

Reagents

BSA (Sigma-Aldrich, A7906)
HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)
EZ-Link Sulfo-NHS-LC-Biotin (ThermoFisher Scientific, A39257)

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 the detection antibody: conjugate belimumab antibody, using the EZ-Link Sulfo-NHS-LC-Biotin.
2. Prepare the unconjugated belimumab capture antibody 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 belimumab, and incubate overnight at 4°C.
3. Wash the microtiter plate five times (5x) with PBST.
4. Block the microtiter plate by adding 100 µL 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
5. Wash the microtiter plate 5x with PBST.
6. For the standard curve, prepare a dilution series of the Anti-Belimumab Antibody HCA429 (AbD60138ia), HCA430 (AbD60136ia), or HCA431(AbD60132ia) in 10% human serum in PBST in triplicate. Final concentration of anti-belimumab antibody should cover the range from 10 ng/mL to 10,000 ng/mL. Include a zero anti-belimumab antibody concentration as the background value.
7. Add 20 µL of anti-belimumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hr at RT.
8. Wash the microtiter plate 5x with PBST.
9. To each well, add 20 µL HRP-conjugated belimumab diluted to 2 µg/mL in HISPEC Assay Diluent and incubate for 1 hr at RT.
10. Wash the microtiter plate 10x with PBST.
11. Add 20 µL QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.