Zhi-Jing Zhao1, Xiu-Mei Liu. 1. National Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing, China.
Abstract
OBJECTIVE: To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E. coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E. coli O157 in foods. METHODS: Spleen cells from BALB/c mice immunized with the somatic antigen of E. coli O157:H7 were fused with murine Sp2/0 myeloma cells. The hybridoma cell line specific for E. coli O157 was established after having been subcloned. Antisera specific for E. coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E. coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. RESULTS: MAb 3A5 specific for E. coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1x10(6). No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1x10(5) with E. coli O157. The detection limit of this sandwich ELISA was 10(3)-10(4) cfu E. coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E. coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. CONCLUSION: MAb 3A5 specific for E. coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E. coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E. coli O157 in food.
OBJECTIVE: To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E. coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E. coli O157 in foods. METHODS: Spleen cells from BALB/c mice immunized with the somatic antigen of E. coli O157:H7 were fused with murineSp2/0 myeloma cells. The hybridoma cell line specific for E. coli O157 was established after having been subcloned. Antisera specific for E. coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E. coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. RESULTS: MAb 3A5 specific for E. coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1x10(6). No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1x10(5) with E. coli O157. The detection limit of this sandwich ELISA was 10(3)-10(4) cfu E. coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E. coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. CONCLUSION: MAb 3A5 specific for E. coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E. coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E. coli O157 in food.