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.