Mouse anti human ige horseradish peroxidase conjugated antibody

The IgE-ELISA assay developed in this study was performed as previously described.³³ (link) Microtiter plates were coated with recombinant polypeptides (200 ng/well) in 0.1 mol/L and pH 9.2 carbonate buffered solution (Leagene, Beijing, China) at 4 °C overnight. The samples were blocked with 300 μl 5% (w/v) Difco™ skim milk (DSM; BD Biosciences) in phosphate-buffered saline containing 0.05% Tween 20 (PBST) at 37 °C for 3 h. Serum samples (1:10 dilution in 1% DSM-PBST) were added to each well (100 μL/well) and incubated for 2.5 h at 37 °C. The plates were incubated with mouse anti-human IgE horseradish peroxidase-conjugated antibody (#9160-05; 1:2000 dilution; Southern Biotech) for 1.5 h at 37 °C. Each incubation step was followed by five washes with PBST. Binding was detected with 100 μl of 1-mM 3,3,5,5′-tetramethylbenzidine substrate (Invitrogen; Thermo Fisher Scientific); the substrate reaction was stopped with 50 μl 2 M H₂SO₄ per well. The plates were read by an absorbance microplate reader (Bio-Rad, USA) at 450 nm. IgE-ELISA results with a positive/negative result sample optical density ratio value > 2.1 were considered positive. All tests were performed in triplicate.