2.4 Measurement of S1-specific IgA, S-IgA and IgG
Two weeks after the last immunization, we measured S1-specific IgA and IgG levels in the serum and S1-specific S-IgA and -IgG levels in BALF by enzyme-linked immunosorbent assay (ELISA). A 96-well plate (Nunc, Naperville, IL) was coated with S1 (0.1 μg/well) in ELISA Coating Buffer (Bethyl Laboratories, Montgomery, TX) overnight at 4°C, then blocked with Tris buffered saline with BSA (pH 8.0) (TBS, Sigma-Aldrich) for 2 h at 37°C. Serum and BALF were added to the plate and 2-fold serially diluted with 0.05% Tween 20-TBS. The plate was washed six times with 50 mM Tris–HCl (pH 8.0) containing 0.14 M NaCl and 0.05% Tween 20 (TTS), incubated with goat anti-mouse IgA or anti-mouse IgG Ab conjugated with HRP (Sigma-Aldrich) for 2 h at 37°C. The plate was washed six times with TTS and incubated with a KPL TMB Microwell Peroxidase Substrate System (SeraCare, Milford, MA) according to the instructions supplied by the manufacturer. The produced chromogen was measured at 450 nm absorbance using a SpectraMax ABS PLUS (Molecular Devices, Sunnyvale, CA). Antibody titers are defined as the reciprocal of the highest dilution of the sample with optical density (OD) of >0.1.