2.2. Measurement of allergen-specific Ig levels
Serum levels of EW-, and OVM-specific IgE were measured using ImmunoCAP (Thermo-Fisher Inc., MA) according to the manufacturer’s instructions. Serum levels of EW-, OVM-, and OVA-specific IgD and IgG4 were determined by ELISA. In brief, microtiter plates (Nalgen Nunc International Co. Tokyo, Japan) were coated with 100 µL/well of EW, OVM, or OVA (100 µg /mL) overnight at 4°C and then were blocked by incubation with ELISA blocking reagent (Roche Diagnostics Co., Tokyo, Japan) for 3 hours at room temperature. After washing four times with PBS/0.05% Tween 20, the plates were incubated with 100 µL/well of diluted serum samples overnight at 4°C. After washing four times with PBS/0.05% Tween 20, plates were then incubated with 100 µL/well of biotin-conjugated rat anti-human IgD or IgG4 antibody (Southern Biotech, AL) at 2 µg/mL in PBS/0.05% Tween 20, followed by washing four times with PBS/0.05% Tween 20 and then a 2-hours incubation with 100 µL/well of horseradish peroxidase-conjugated streptavidin (X4000 in PBS/0.05% Tween 20, Thermo-Fisher Inc.). The plates were developed with the TMB microwell peroxidase substrate system (Kirkegaard and Perry Laboratories Inc., Guildford, UK). The antigen-specific antibody titers in the serum were expressed as arbitrary units determined using the standard pooled serum, which contained high titers of antigen-specific antibody.