IgA and IgG Quantification of Anti-Flagellin

Quantification of anti-flagellin- specific IgA and IgG has been previously described^{32 (link)–34 (link)}. In brief, 96-well microtiter plates (Costar, Corning, New York) were coated with 100 ng/well of either laboratory-made Salmonella Typhimurium-derived or purchased B. subtilis-derived flagellin in 9.6 pH bicarbonate buffer overnight at 4 °C. Serum or fecal samples from mice were then applied either pure or at a 1:100 dilution for 1 h at 37 °C. After incubation and washing, the wells were incubated with either horseradish peroxidase-linked anti-mouse IgG (GE Healthcare Life Sciences, NA931V) or horseradish peroxidase-linked anti-mouse IgA (Southern Biotech, 1040–05). Quantification of immunoglobulin was then developed by the addition of 3,3′,5,5′-Tetramethylbenzidine and the optical density was calculated by the difference between readings at 450 nm and 540 nm.