Enzyme-linked immunosorbent assay (ELISA)
Purified fish allergens diluted in coating buffer (100 mM Na2CO3. 100 mM NaHCO3, pH 9.6) were coated onto MaxiSorp microtiter plates (Nunc) and incubated overnight at 4°C. After washing the plates with 0.05% Tween-20/PBS (PBS-T) and blocking the plates with 5% fetal bovine serum (Gibco) diluted in PBS (blocking buffer) at room temperature for 2 hours, serum samples diluted at 1:10 in blocking buffer were added for overnight incubation at 4°C. Intensity of IgE binding was detected by incubating the plates with biotinylated anti-human IgE antibodies (1:1000 dilution, Vector Labs), HRP avidin D (1:1000 dilution, Vector Labs) and TMB substrate (BD Biosciences). Upon terminating the reaction with 0.1M sulfuric acid, the optical density (OD) at 450 nm was measured using a microplate reader (BioTek). Results were considered positive only if the OD is 3-fold higher than the mean of non-atopic negative controls.