Preparation Of Discriminatory Monoclonal Antibodies For Detection Of E.coli O157:H7 Using Subtractive Immunization

Outbreaks and prevalence of diseases caused by E. coli O157: H7 have been threatening severely to human health. Especially the highly pathogenic, low-dose infection and the long incubation period result in great difficulty in diagnosis. Therefore, it is necessary to develop rapid, sensitive and specific detection methods to ensure food safety.At present, polyclonal antibody is usd for the most immunological detection of E. coli O157:H7. The polyclonal antibody of E. coli O157:H7 has serious cross-reaction with other intestinal bacteria, which can not meet the precise needs of serological testing.The reports on preparation of monoclonal antibodies (McAbs) against E. coli O157:H7 are rare. To preparation of discriminatory monoclonal antibodies to E. coli O157:H7 surface molecules, the main problem is difficult to screening. The valuable antibodies can not be got directional.In this study, hybridoma cell lines which secreted monoclonal antibody specific against E. coli O157: H7 was prepared and identified. It is suggested that the McAbs had the potential to be used in developing a rapid, easy and practical immune colloidal gold filtration assay for detection of E. coli O157: H7 from a wide range of bacteria. 1. Preparation of McAbs: the splenocyte of BALB/c mice immunized with subtractive immunization were fused with SP2/0 myeloma cells. Nineteen strains McAbs to E. coli O157: H7 were established after screening by indirect ELISA and subcloning by limiting-dilution, which were named as 1 C6, 1D8, 1H6, 3F5, 3G12, 4A7, 4D7, 5A2, 5A5, 5A9, 5C12, 5D8, 5F10, 5F11, 5H4 , 6C11, 6D12, 7F1and 7F8 ,respectively. Hybridoma chromosome analysis showed the hybridoma cell lines had 96-105chromosomes, about the chromosomes number of the plus of mice spleen cells and SP2/0 myeloma cells.2. Identification of McAbs: 19 McAbs measured by indirect ELISA with 81 strains detection bacteria to observe the cross-reaction situation. 1C6, 1D8, 1H6, 4A7, 5A2 and 5D8 six hybridoma cell strains secreted specific McAbs for E. coli O157: H7 and have a good antibody response spectrum with four field strains of E. coli O157: H7. Dot-ELISA had a similar result. The immunoglobulin subclasses of 1C6, 1D8, 4A7, 5A2 and 5D8 were IgM, IgG1, IgG2b, IgM and IgM, respectively. The affinity constants of 1C6, 1D8, 4A7, 5A2,5D8 against E. coli O157: H7 were1.90×1010, 1.57×1010, 2.79×1010, 1.47×1011 and 1.83×1010(mol/L)-1,rerspectively. The ELISA titers of culture supernatant and ascites were about 1:103 and 1:106. The result of western blotting revealed that the McAbs from 1C6, 1D8, 4A7, 5A2 and 5D8 could react with cell protein of E. coli O157: H7.3. Application of the McAbs: the McAbs of 4A7 and 5A2 from ascites were purified, and tagged with colloidal gold. Then the dot immuno-gold filtration assay was developed to examine E. coli O157: H7. The results of dot immuno-gold filtration assay showed that the positive outcome was gained with E. coli O157: H7 and there was no cross-reaction with 74 non-O157 strains. The minimum detection limitation of E. coli O157:H7 bacteria suspension was 5.5×106cfu/mL.The sensitivity of detection can be increased by one order of magnitude by addition experiment with 4A7 tagged with colloidal gold or silver staining. The minimum detection limitation of E.coli O157:H7 bacteria suspension reached 5.5×104cfu/mL by using immunomagnetic separation and silver staining.The preparation and preliminary application of the McAb anti- E. coli O157: H7 will contribute to the further improvement of the detection of E. coli O157: H7 and to provide a useful tool and assay for diagnosis of E. coli O157: H7.