Development Of Monoclonal Antibody Against Enterohemorrhagic E. Coli O157:H7and Immunochromatographic Strip For The Detection

Enterohemorrhagic Escherichia coli (EHEC) O157:H7is one of the significant foodborne pathogen recognized in1982during an outbreak of severe bloody diarrhea in USA. It can lead to hemorrhagic or nonhemorrahagic diarrhea and other diseases such as hemorrhagic enteritis (HC), hemolytic-uremic syndrome (HUS). thrombotic thrombocytopenic purpura (TTP) in human and animals.EHEC O157:H7has been found in the intestines of healthy cattle, deer, goats and sheep. Ruminants are principal reservoir. Because there is no specific treatment available for the disease currently, there is an urgent need for effective preventive measures. Such measures will be dependent on the availability of rapid, sensitive and simple procedures for the detection of the pathogen. Classical bacteriological isolation method is golden standard, but it is tedious and time costing. Immunological methods and molecular biology methods for identification of EHEC O157:H7are getting more and more important.There are three patrs in this study.Part one:Five hybridomas cell lines secreting monoclonal antibodies (MAbs) against enterohemahagic Escherichia coli (EHEC) O157:H7were established by fusing SP2/0with spleen cells from BALB/c mice immunized with formaldehyde-killed E. coli O157:H7, naming1B1,2D6,2D9,2E3and2G5. The Ig isotypes of2G5was IgG3, and the others were IgM. Light chain were all the κ. The indirect ELISA titers of the ascites were1×105,1×104,1×106,1×107and1×107respectively. The result of agglutination test showed that these MAbs did not react with other strains, such as EHEC O26:H11, EHEC0111and so on, and possessed high degree of specificity.Part two:The gold particle in colloidal solution was coupled with purified monoclonal antibody2G5, and then sprayed on the glass fibers. The purified monoclonal antibody2E3served as the capture-antibody was coated on the nitrocellulose membrane. Then the sandwich-based gold immunochromatographic (IC) strip for the detection of E. coli O157:H7was assembled in regular order. The detection results indicated that the IC strip was specific to O157:H7and the sensitivity was106CFU/ml for detecting sample. The IC strip is more convenient, rapid and simple for the preclinical diagnosis and field investigation of E. coli O157:H7infection or contamination.Part three:The mice were inoculated intragastrically of Escherichia coli O157:H7, it was found that O157:H7in faeces could be detected on hour2after inoculation in inoculated mice by using the combined methods of both immunochromatographic (IC) strip and plate culture count with differential medium of E. coli O157:H7and the peak levels of O157:H7in faeces were attained during the sixth hour after inoculation and then it dropped down gradually up to day15following inoculation. The result of the two methods is identical, but the IC strip is convenient and rapid for the clinical diagnosis and field investigation of E. coli O157:H7infection or/and contamination. The research provided animal models and detection methods for epidemiology, pathopoiesis mechanism and control of E. coli O157:H7.