Rme 1

For measuring total IgE in serum, an enzyme-linked immunosorbent assay (ELISA) plate was coated with purified monoclonal anti-mouse IgE antibody (RME-1, BioLegend) for 2 hours at room temperature (RT). Then, the plate was incubated with blocking buffer [5% FBS in phosphate-buffered saline (PBS)] for 1 hour. After blocking, the plate was washed, and diluted sera and standards were incubated for 2 hours at RT. Horseradish peroxidase–conjugated polyclonal anti-mouse IgE (Southern Biotech) was used as a detection antibody. After a 2-hour incubation with a detection antibody, peroxidase reaction was visualized by adding tetramethylbenzidine (TMB) substrate solution (SurModics). Plates were read at 405 nm using a spectrophotometer. For measuring food Ag–specific IgE, NCD was grounded and incubated overnight in PBS to solubilize. Insoluble fraction was removed by filtering and centrifugation. Soluble fraction was collected and used as the diet extract. Protein concentration of soluble fraction was measured by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Diet extract (0.5 mg/ml) and control protein (OVA) were coated on an ELISA plate. To enhance the detection of Ag-specific IgE in sera, IgG in sera was depleted by coincubation of protein A/G (GE Healthcare). One-fifth diluted sera were incubated overnight at 4°C, and ELISA was performed as described above.

Peripheral (inguinal, axillary, and cervical) lymph nodes, spleen, MLNs, and PPs in GF or AF mice were collected, and single-cell suspension was prepared. To enrich plasmablasts, lymphocytes were depleted by using biotinylated anti-B220 and anti-CD90.2 antibody and streptavidin-conjugated magnetic beads (BD Biosciences). Enriched cells (0.1 × 10⁶ to 0.5 × 10⁶) were added on an ELISPOT plate previously coated with anti-mouse IgE (RME-1, BioLegend). After 8 hours of incubation at 37°C, the plate was washed with PBS containing 2% Tween 20 and incubated with biotinylated monoclonal anti-mouse IgE (JKS-6, BioLegend) followed by streptavidin-conjugated alkaline phosphatase (BD Biosciences). Spots for IgE-producing cells were detected by addition of the bromochloroindolyl phosphate–nitro blue tetrazolium (BCIP-NBT) substrate (Sigma-Aldrich) solution.