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.