| Enterohaemorrhagic E.coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance causing foodborne infections with possibly life-threatening consequences in humans, such as haemorrhagic colitis(HC), haemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenia purpura (TIP).EHEC O157:H7 is an zoonotic bacteria, Ruminants (mainly cattle) are considered a primary reservoir and the pathogen has been isolated from stool specimens of pigs, sheep, horses, dogs, geese, rabbits, chickens, fish, deer and other animals. Occasionally, EHEC O157:H7 has been associated with diarrhoea in young calves and contamition of headwater, vegatables, fruits, dairy products, consituite a great threat to people’s health. Antibiotic will promote the rapid release of Shiga toxin and accelerate deterioration of the patient infected with EHEC O157:H7. Therefore, it is impossible for human to get immunity and resistance EHEC 0157:H7 infections.Establishing and improving the detection means for EHEC 0157:H7 is essential for prevention of this disease. Intimin, a 94-kDa outer membrane protein, mediates the adhesion of enteropathogenic Escherichia coli(EPEC) and enterohemorrhagic Escherichia coli (EHEC) to enterocytes. Intimin is encoded by the E. coli attaching and effacing (eae) gene, The variable 280-amino acid C-terminal sequence of intimin (Int280) defines many different intimin subtypes, has favorable immunogenicity and protective immunity. As the superiority of monoclonal antibodies and ELISA, we prepared the monoclonal antibody against intimin and established a double sandwich ELISA for detecting EHEC O157:H7. The results are followed:1 Generation the MAbs against intiminIn this experiment, hybridomas were established by fusing SP2/0 with spleen cells of mice immunized with purified intinimin when the titer above 104. The positive wells were selected by indirect ELISA, and after 4 generations of clone by limited dilution, two hybridized cell clones(2B10,4D7)against intimin were obtained. The titers of the ascetic were 5.2×104 and 2.5×104 respectively and the MAbs were specific to EHEC O157:H7 and not reacted with E.coli O139、E.coli K88、EHEC O26:H11. Western-blot analysis showed that, two monoclonal antibodies can react with the fusion protein and detect EHEC O157:H7 specifically. Indirect ELISA shows the limitation of two MAbs for intimin were 75 ng/ml.2 Establishment of double antibody sandwich ELISA for detecting EHEC O157:H7Rabbit anti-EHEC O157:H7 serum was isolated from rabbit immunized with inactivated EHEC O157:H7 and then purified. And the double sandwich ELISA method for detecting EHEC O157:H7 was established by using the monoclonal antibody against intimin and the purified serum. The best working concentration of purified serum and anti-intimin McAb were 3.2 μg/ml and 4.75 μg/ml, respectively. The best blocking reagent was 2%BSA and the optimal blocking time was 1 h. The reaction time for capturing antigen was 1 h and The reaction time for HRP-labled antibody was 45 min. The reaction time for TMB was 7 min. The ELISA method developed in this present study possesses high specificity and repeatability, doesn’t cross-react with E.coli 0139, E.coli K88, Shigella and Sramana. Limitation of the method for detection EHEC O157:H7 were 1×105 CFU/ml. |
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