Allergen-specific serum IgE measurement
Allergen-specific serum IgE levels were measured by ELISA. Briefly,
wells of microtiter plates (Costar, Corning Inc., Corning, NY, USA) were
coated with 100 μL/well of HSE (500 μg/mL) and reference wells were
coated with purified anti-mouse IgE capture antibody (BD Biosciences
Pharmingen, San Diego, CA, USA). The next day, the plates were blocked
and washed, and then 50 μL of each serum sample (diluted 1:5 in blocking
buffer) was applied to sample wells. A series of eight two-fold
dilutions of purified mouse IgE (BD Biosciences Pharmingen) were used in
conjunction with reference wells as standards; plates were then
incubated overnight. Subsequently, they were treated with 100 μL of
biotinylated monoclonal anti-mouse IgE antibody (1 μg/mL, BD Biosciences
Pharmingen) and horseradish peroxidase-streptavidin (diluted 1:1,000; BD
Biosciences Pharmingen) for 1 h each at 37°C. Finally, 100 μL/well of TM
Blue (Cwbiotech, Beijing, China) was applied as a substrate, and the
reactions were allowed to develop at room temperature for 20 min. The
plates were read at 450 nm using an ELISA plate reader (BioTek, ELX800,
Winooski, VT, USA). A standard curve
of murine IgE was used as a reference.