Goat poly

A mouse IgG ELISA Quantitation Kit (Bethyl Laboratories, TX, USA) was used according to the manufacturer’s instructions. In brief, the immobilized antibody (goat-poly, anti-mouse IgG, Bethyl Laboratories) 10 µg/mL diluted with a carbonate buffer (0.05 M, pH 9.6) was added to a 96-well ELISA plate at 100 µL/well and incubated at 25 °C for 1 h. It was then washed five times with ELISA buffer (50 mM Tris, 140 mM NaCl, 0.05% Tween20, pH 8.0) and blocked with 1% bovine serum albumin for 1 h. Next, 100 µL of the target antibody (mouse, anti-goat IgG, #105-3102, Rockland Immunochemicals, PA, USA) was diluted with Blocking One (Nacalai Tesque, Kyoto, Japan) and added after washing five times with ELISA buffer for 1 h. A concentration of 0.0273 to 3.50 µg/mL was used as the standard solution for the calibration curve. After washing five times, 100 µL of labeled antibody (goat-poly, anti-mouse IgG-HRP, Bethyl Laboratories) 10 ng/mL diluted with ELISA buffer was added for 1 h. After washing five times, the samples were developed using the ELISA POD Substrate TMB kit (Nacalai Tesque, Kyoto, Japan) at 25 °C for 30 min. Subsequently, quenching with sulfuric acid (0.18 M), the absorbance was measured at 450 nm.

Total IgG1, IgG2a, IgG2c levels were determined using an ELISA Quantitation Set (Bethyl). IgE and OVA-specific IgE levels were measured with an ELISA MAX Standard Set (Biolegend) and DS Mouse IgE ELISA (OVA) (DS Pharma Biomedical), respectively. For OVA-specific IgG1, IgG2a and IgG2c detection, serial diluted sera were sandwiched between plate-coated 1.0 μg OVA and horseradish peroxidase-conjugated anti-mouse IgG1 (Rat-mono, LO-MG1-2; AbD Serotec), IgG2a (Rabbit-poly; Invitrogen) or IgG2c antibodies (Goat-poly; Bethyl). The reactions were developed with 3, 3', 5, 5'- tetramethylbenzidine (TMB; KPL), and the absorbance was read by plate reader. Anti-ovalbumin IgG1 antibody (mouse-mono, 8C6; Thermo) and anti-denatured ovalbumin IgG2a antibody (mouse-mono, 6G2; Thermo) were used as reference antibodies. The relative concentration of OVA-specific IgG2c was compared by absorbance, because no anti-ovalbumin IgG2c antibody could be obtained.