Allergen-specific serum IgG2a measurements
Allergen-specific serum IgG2a levels were measured by ELISA. Briefly, plates were coated with 100 μL of HSE (5 μg/mL) overnight at 4°C and reference wells were coated with anti-mouse IgG2a (0.5 μg/mL, BD Biosciences Pharmingen). After the wells had been blocked and washed, serum (diluted 1:1,000) was added to HSE-coated wells and purified IgG2a (BD Biosciences Pharmingen) was added to reference wells; the plates were incubated overnight at 4°C.Plates were subsequently washed and incubated with alkaline phosphatase-labelled goat anti-mouse IgG2a (diluted 1:2,000;Southern Biotechnology Associates, Birmingham, AL, USA) for 2 h at room temperature. After plates had been washed, a solution of 4-nitrophenyl phosphate (Roche, Mannheim, Germany) was added to each well. Absorbance at 450 nm was measured 1 h after the addition of substrate. A standard curve of murine IgG2a was used as a reference.