Purified rat anti mouse ige

To measure OVA-specific IgE levels in plasma samples, 96 well half-area plates were coated with 0.5 mg/mL purified rat anti-mouse IgE (BD Biosciences, NJ) in PBS and incubated for 2 h at room temperature. After washing, the plates were incubated with 0.1x Block Ace (KAC, Kyoto, Japan) for 1 h at room temperature. The plates were then incubated with standard or diluted plasma overnight at 4°C. After washing, Ovalbumin HRP (Bio-Rad, CA) was added to the plates. One hour later, TMB Microwell Peroxidase Substrate System (KPL) was added to the wells. After incubation for 20 min, the reaction was stopped by adding 2 N H₂SO₄. The absorbance at OD450 nm was measured and the concentration of OVA-specific IgE in plasma was calculated according to the standard. For analyses by machine learning algorithms, the concentrations of IgE of each herbal medicine extract group and each control adjuvant group, from which the concentration of OVA alone group was subtracted, were used.

Albumin from chicken egg white (OVA), fetal bovine serum (FBS), penicillin, Roswell Park Memorial Institute 1640 (RPMI 1640) medium, and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purified rat anti-mouse IgE, biotin rat anti-mouse IgE, goat affinity purified antibody to mouse IgG, and biotin-XX goat anti-mouse IgG₁ were purchased from BD Biosciences (Franklin Lakes, NJ, USA), MP Biomedicals (Santa Ana, CA, USA), and Life Technologies (Carlsbad, CA, USA), respectively.