Goat anti mouse ige

The quantification of anti‐OVA‐specific IgE in the sera of mice was carried out by enzyme‐linked immunosorbent assay (ELISA). Briefly, the ELISA plates (Costar) were coated with 100 µL/well OVA (100 µg/mL) in pH 9.6 carbonate‐bicarbonate buffer at 4°C overnight and then blocked for 2 hours with 200 µL/well skimmed milk powder (5%). After washing, the diluted sera were added and incubated for 2 hours at 37°C. After washing, 1:250 dilution of goat anti‐mouse IgE (Abcam) was added and incubated for 2 hours at 37°C. After removing the unbound antibodies, HRP‐conjugated rabbit anti‐goat secondary IgG (1:5000, Multisciences) was incubated for 1 hours at 37°C. The colour reaction was developed by adding 100 µL/well of TMB solution (eBioscience) for 15 minutes and then stopped with 50 µL/well of 2 M sulphuric acid. Finally, it was read at 450 nm in an ELISA reader (Bio‐Rad).

Polystyrene plates were coated with ovalbumin solution at 5 μg/mL in carbonated coating buffer at 4°C overnight. On the next day, the plates were washed (PBS 1X+0.05% Tween®20) and blocked (PBS 1X+0.25% casein) at room temperature for 2 hours. For IgG detection, the serum samples were diluted at 1 : 1000 and BAL at 1 : 20. For IgA, the serum samples were diluted at 1 : 10 and BAL at 1 : 20. For IgE, both serum and BAL samples were diluted at 1 : 20. Samples were incubated at 4°C overnight. Plates were then washed and incubated with biotinylated anti-mouse IgG1 (Thermo-Scientific, USA), IgA (eBioscience, USA), or goat anti-mouse IgE (Abcam, USA) antibodies for 1 hour at room temperature. Plates were washed and incubated with streptavidin-conjugated to peroxidase (IgG and IgA) (Sigma-Aldrich), for 1 hour at room temperature. The reaction used OPD and was stopped with sulfuric acid. Colorimetric absorbances were measured at 492 nm.