Antibody concentrations in collected blood and supernatant were determined using IgE (BioLegend 432401), IgM (Thermo Fisher Scientific 88-50470-22), IgG1 (Bethyl Laboratories E90-105), IgG2a (Bethyl Laboratories E99-107), and IgG2b (Bethyl Laboratories E99-109) ELISA kits. Freshly collected blood samples were prepped for ELISA by first allowing samples to clot and then centrifuging at 1500g for 15 minutes. The clear plasma, not including the buffy coat, was collected and frozen (–20°C) for subsequent ELISA. The serum samples were diluted at 1:50 for IgE ELISAs or 1:1000 for IgG1, IgG2a, IgG2b, and IgM ELISAs in the respective kit sample diluent. Supernatant collected from in vitro cultures was used undiluted for the IgE ELISA and diluted 1:5 for all other ELISAs. Differences in ELISA dilutions were equalized prior to analysis. Kit protocols were followed for standards and addition of capture antibody, detection antibody, TMB, and stop solution (2N sulfuric acid). For OVA-specific ELISAs, 96-well ELISA plates were instead coated with 1 μg/mL OVA (MilliporeSigma) and incubated overnight at 4°C, and then kit protocols were followed (61 (link)). Absorbance was measured at 450 nm wavelength.
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