Quantifying Anti-Salmonella Antibodies by ELISA

These experiments were performed as previously described (25 (link)). Briefly, enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immuno) were coated overnight with 100 μl of carbonate-bicarbonate buffer (Sigma-Aldrich) per well containing 7.5 μg/ml S. Typhimurium LPS antigen (Alexis Biochemicals). Plates were washed with PBS containing 0.05% Tween 20 and blocked with 200 μl/well blocking buffer (PBS–1% bovine serum albumin [BSA]) for 1 h at 37°C. Test serum prepared at 1:20 in dilution buffer (PBS–0.05% Tween 20–1% BSA) was serially diluted 3-fold and incubated at 37°C for 1 h. After washing, 100 μl of 1:2,000 secondary goat anti-human IgG-AP antibodies (Southern Biotech) was added and incubated for 1 h at 37°C. Finally, after washing, 100 μl of SigmaFAST p-nitrophenyl phosphate substrate was added to each plate and the plate was read after 30 min using a BioTek ELx800 reader (BioTek Instruments, USA) at 405 nm.