Isotype-specific detection of immunoglobulins (Igs) was carried out according to the protocol by Germundson and Nagamoto-Combs [49 (link)] with modifications. Eight-well RIA strips (Corning, Inc., Corning, NY, USA) were coated with 2 μg/mL BLG in a sodium carbonate/bicarbonate buffer (pH 9.5) overnight at 4 °C. The wells were washed and blocked in the PBS containing 0.5% bovine serum albumin (BSA). The serum samples were diluted to 1:40 and incubated with protein G-coated plates (Thermo Fisher, Waltham, MA, USA) for 1 h at 37 °C to adsorb total IgG, and the resulting supernatant was subsequently added to each well. Allergen-specific IgE was detected using secondary anti-mouse IgE and avidin HRP (eBioscience, San Diego, CA, USA) [49 (link)]. The substrate reaction was terminated with 2N sulfuric acid, and the plates were immediately read at 450 nm with a reference wavelength of 550 nm on an ELx800 Universal Microplate Reader (BioTek Instruments, Winooski, VT, USA).
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