Horseradish peroxidase

To confirm the sensitization to OVA or ω5-gliadin, the plasma levels of IgE Abs specific to OVA or ω5-gliadin were determined using ELISA. Briefly, the wells of the F8 MaxiSorp Loose Nunc-Immuno™ Modules (Thermo Fisher Scientific) were coated with 100 μl of OVA (10 μg/ml) dissolved in phosphate buffered saline (PBS) or ω5-gliadin (20 μg/ml) dissolved in 0.1% acetic acid overnight at 4 °C. After washing with phosphate buffered saline containing 0.1% Tween 20 (PBS-T) six times, plates were incubated with 1% Block Ace® (DS Pharma Biomedical Osaka, Japan) for 2 h at room temperature. Then, 100 μl of each sample of rat plasma (diluted 1:10 in 1% Block Ace®) was added to each well and incubated for 2 h at room temperature. After washing with PBS-T, the wells were incubated with 100 μl of horseradish peroxidase (HRP)-conjugated mouse anti-rat IgE Ab (GeneTex, Irvine, CA, USA) (diluted 1:1000 in PBS) for 2 h at room temperature. The wells were washed with PBS-T and then incubated with 100 μl of 3,3′,5,5′-tetramethylbenzidine solution (KPL, Gaithersburg, MD, USA) at room temperature. After 15 min incubation, the reaction was terminated with 100 μl of 1 mol/l phosphoric acid. Absorbance was measured at 450 nm against 630 nm as a reference using a Multiskan GO (Thermo Fisher Scientific).