Protocol: ADA IgE Isotype ELISA  For Use With Anti-Adalimumab Antibody

Reagents

BSA

HISPEC immunoassay diluent (Bio-Rad, BUF049)

Human Serum (Sigma-Aldrich, H4522)

Mouse anti-human IgE antibody, HRP conjugated (Bio-Rad, 0100-0413P)

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 adalimumab 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 adalimumab, 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 the anti-adalimumab antibody HCA271 (AbD18655_hIgE) in 10% human serum in PBST in triplicate. Final concentration of anti-adalimumab antibody should cover the range from 0.05 ng/ml to 2,000 ng/ml. Include a zero anti-adalimumab concentration as the background value.
6. Add 20 µl of anti-adalimumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
7. Wash the microtiter plate five times with PBST.
8. To each well add 20 µl HRP labeled mouse anti-human IgE antibody diluted 1:1,000 in HISPEC buffer and incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. Add 20 µl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V1.11.2016