2.8. Quantitative enzyme-linked immunosorbent assay (ELISA)

S. Typhimurium LPS was purchased from Sigma (St. Louis, MO, USA). Salmonella outer membrane proteins (SOMPs) were prepared as previously described in [26]. A quantitative ELISA was used for the analysis of antibodies in serum to S. Typhimurium LPS and SOMPs. The wells of polystyrene 96-well flat-bottom microtiter plates were coated with 100 ng/well LPS or SOMPs suspended in 100-µl volumes of sodium carbonate/bicarbonate coating buffer (50 mM Na₂CO₃, 50 mM NaHCO₃, 0.1% sodium azide, pH 9.6). Goat anti-mouse Ig (H + L) (BD Pharmingen; San Diego, CA) in PBS was added to extra wells without coating with OMPs or LPS in triplicate to determine the standard curve. The coated plates were incubated overnight at 4 °C, followed by blocking with PBS containing 10% fetal bovine serum (FBS) for 1 h at room temperature. A 100-µl volume of 200-fold diluted sample was added to each corresponding well in triplicate, and 100-µl volume of the mouse IgG (BD Pharmingen; San Diego, CA, USA) with twofold dilutions in PBS from 0.5 mg/ml was successively added to the well coated by goat anti-mouse Ig (H + L) for the standard curve. The plates were incubated for 1 h at 37 °C and were then treated with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates; Birmingham, AL, USA). The wells were developed with a streptavidin-alkaline phosphatase conjugate (Southern Biotechnology Associates; Birmingham, AL, USA), followed by p-nitrophenylphosphate substrate (Sigma-Aldrich; St. Louis, MO, USA) in diethanolamine buffer (pH 9.8). Color development (absorbance) was recorded at 405 nm using an iMark™ Microplate Reader (Bio-Rad; Hercules, CA, USA). The standard curve was drawn using Curve Expert (Hyams DG; Starkville, MS, USA), and the concentration of serum antibodies was calculated using the standard curve.