The total IgE and HDM-specific IgE levels were measured in the serum of HDM-sensitized mice. Total IgE levels were measured using an ELISA MAX Deluxe Set (BioLegend). To measure HDM-specific IgE levels, 96-well plates (Greiner Bio One, Kremsmunster, Austria) were coated with 50 µg/mL HDM extract in coating buffer (Bio Rad, Hercules, CA, USA) and incubated overnight at 4 °C. These plates were blocked with Blocking One (Nacalai Tesque, Osaka, Japan) overnight. Next, serum samples were added to these plates and incubated for 2 h at 37 °C. After washing, the cells were treated with biotinylated monoclonal antibody against mouse IgE (YAMASA, Chiba, Japan) and incubated for 1 h at 37 °C. After washing, horseradish peroxidase (HRP)—conjugated streptavidin (BioLegend) was added and incubated for 1 h at 37 °C. The serum of the mice injected intraperitoneally with HDM and Imject Alum Adjuvant (Thermo Fisher Scientific) was used to prepare the standard and diluted with Solution A (Nacalai Tesque). For serum IgE capture assay, 96-well half plates (Greiner Bio One) were coated with 2 µg/mL purified monoclonal antibody against mouse IgE (YAMASA) in coating buffer (Bio Rad) and incubated overnight at 4 °C. These plates were blocked with Blocking One (Nacalai Tesque) for 1 h at 37 °C. Next, serum samples were added to these plates and incubated overnight at 4 °C. After washing, the plates were treated with 10 µg/mL biotinylated HDM extract and incubated for 1 h at 37 °C. After washing, horseradish peroxidase (HRP)—conjugated streptavidin (BioLegend) was added and incubated for 30 min at 37 °C. The serum of the mice injected intraperitoneally with HDM and Imject Alum Adjuvant (Thermo Fisher Scientific) was used to prepare the standard and diluted with Solution A (Nacalai Tesque). IL-4 and IL-13 levels in the supernatant of MLN cells stimulated by HDM (detailed below) were measured using a mouse IL-4 Quantikine ELISA kit (R&D systems, Minneapolis, USA) and mouse IL-13 DuoSet ELISA (R&D Systems). A plate reader (TECAN, Mannedorf, Switzerland) was used to measure the absorbance of each well at 450 nm.

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