Biotinylated anti ige

For serum OVA-specific IgG1 antibody detection, Nunc Maxisorp 96-well plates (Thermo Fisher Scientific) were coated with 10 μg/ml OVA (Grade V, Sigma-Aldrich) O/N at 4°C, subsequently blocked with 1 % BSA and serial dilutions of sera were added (1:1000, 1:2500, 1:5000, 1:10000). Purified mouse IgG1 (BD Biosciences) was used as standard (starting concentration 500 ng/ml). Detection was achieved with biotinylated anti-IgG1 (BD Biosciences) at 0.1 μg/ml. For total IgE detection, the plates were coated with anti-mouse IgE (BD Biosciences) at 2 μg/ml and antibody was detected with biotinylated anti-IgE (BD biosciences) at 0.1 μg/ml. Purified mouse IgE (BD Biosciences) was used as standard (starting concentration 500 ng/ml). For OVA-specific IgE detection, the plates were coated with anti-mouse IgE (BD Biosciences) as above and the antibody was detected by biotin-OVA (Nanocs) at 10 μg/ml. Mouse anti-OVA IgE (Bio-Rad was used as a standard (starting concentration 250 ng/ml). Antibody binding was detected with streptavidin-HRP (BD Biosciences) and developed with TMB substrate set (BD Biosciences).