ELISA for Gut Immunoglobulins

Immunoglobulins in the culture supernatants or luminal gut were measured by direct ELISA. Briefly, samples or standards were used to coat the 96-well plate overnight at 4°C. After washing and blocking (1-hr, RT) with 1% BSA in PBS on a shaking platform (400 rpm), the samples were then incubated with AP–conjugated goat anti-mouse IgA, IgG or IgM (Southern Biotech, Birmingham, AL, USA) for 2-hrs RT on a shaking platform). Samples were subsequently washed and PNPP substrate (Sigma-Aldrich) was added. The reaction was stopped upon addition of 1M NaOH. Samples were analyzed on a microplate spectrophotometer (Perkin Elmer) at 405 nm (OD). Antibody concentrations were determined by linear regression of standard curves.