Pig screen elisa

Purified recombinant proteins in 0.05 M sodium carbonate-bicarbonate buffer (pH 9.6) were coated (4 °C overnight) on microtiter plates (MaxiSorp, Nunc, Denmark) that were blocked afterwards with 1 % casein hydrolysate in PBS/T (30 min). The plates were subsequently incubated (one hour, RT) with 100× diluted porcine sera, washed with PBS/T and then incubated (one hour, RT) with goat anti-pig IgG conjugated with HRP (Bethyl Laboratories, USA). After a washing step, the colour was developed by adding the chromogenic substrate TMB Complete (Test-line, Czech Republic) and the reaction was stopped with 2 M sulfuric acid (Penta, Czech Republic). The absorbance was measured at 450 nm on Synergy H1 (Biotek, USA). IgG antibody response to LPS was determined by commercially available Pig Screen ELISA (Qiagen, Germany). The individual serum samples were tested in duplicate by ELISA. Positive thresholds in time course graphs were counted as an average plus three standard deviations of all control samples and time zero samples of respective groups [11 ]. Positive thresholds for LPS commercial kit were provided by a manufacturer.

Three groups of animals consisting of twelve white mixed-breed piglets (bought from a commercial stud) weaned 21 days after birth were used in the experiment. Pigs in the first group remained serologically negative for anti-Salmonella antibodies (determined by Pig Screen ELISA, Qiagen, Germany). Another twelve animals were orally infected one week after housing with 1 × 10⁸ CFU of Salmonella Typhimurium grown in BHI medium and blood was collected 28 days after the infection. The last group of animals was vaccinated intramuscularly into the neck with 1 ml of a vaccine prepared from 1 × 10⁹ CFU of Salmonella Typhimurium grown in BHI medium, inactivated with formaldehyde and adjuvanted with Montanide ISA50V2 (Seppic, France). The first dose was administered one week after housing and the second dose two weeks later. Blood was collected 14 days after the second dose of a vaccine.