Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food

Abstract

Salmonella is one of the most important food-borne pathogens. Early detection of Salmonella is necessary for food safety and hygiene. We have developed an ELISA assay, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant flagellar antigens or H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, α cluster and G complex (McQuiston, J. R. et al., 2004). The genes coding for antigens (fliCgm and fliCi) were cloned into vector pGEX-5X-1 and expressed in the strain E. coli BL21(DE3). The recombinant E. coli was cultured in 200ml LB and the expression of fliCgm and fliCi was induced with 0.4mM IPTG. 2.5mg of each GST-fused protein (GST-H:g,m and GST-H:i) was harvested after purifying by GSTrap FF column. The protein was confirmed by SDS-PAGE analysis and Western Blot with the specific anti-H antigen antibody. 100μg of protein in complete Freund adjuvant was injected to rabbit. Blood collection and booster injection were done every 3 weeks. Terminal blood collection was at the fourth booster injection. 100ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP. The sandwich ELISA procedure utilized the mixture of antibodies. After optimization, the concentration of the mixture of antibodies in the sandwich ELISA method was 250ng of capture antibodies and 100ng of antibody-HRP conjugate. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples was positive of Salmonella presence (OD492>0.3), and only 1% was negative (OD492<0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). In conclusion, the ELISA assay was confirmed the specificity to Salmonella sp. and a very low cross-reactivity to other bacteria presenting in food.