Preparation And Identification Of Monoclonal Antibodies Against Enterorrhagia Escherichia Coli O157:H7

Enterorrhagia Escherichia coli (EHEC)is one of the foodbome pathogenic bacteria which was discovered in recent years,and O157:H7 is the major serotype. EHEC can lead to severe diseases such as hemorrhagic enteritis (HC), thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS) and so on in human and animals. In the old and the young, the death mortality of hemorrhagic enteritis was 30 percent higher, and that of HUS was even more than 50 percent. At present ,there are not effective method against the infection of O157:H7 except conservative therapy and antitbiotic therapy.However, studies showed most of the antibiotics can promote or induce the bacterium to release Shiga toxin , thus the HUS was be provoked.In order to control the outbreak or the prevalence of the food poisoning and bacteria infection of O157:H7, accurate and fast detection is the key point.Too many ways are supplied for identifying this pathogen, but only when the biochemistry reaction, serologic test and virulence factor test were undertaken can the bacterium be confirmed according to international standard. Quick-detecting method need to be established for the test procedure is time consuming. Cell-fusion was used to preparing the monoclonal antibodies (McAbs) against EHEC O157:H7, and specificity identification of the McAbs were taken. These were the good foundation for preparing colloidal gold immunochromatography test papers.1. Preparation of McAbThe entire bacterium was used as antigen to immunize eight-weeks-old BALB/C mice with interval of three weeks.Three days after booster, SP2/O myeloma cells and spleen cells of the immunized mouse were fused by PEG-1000.The hybridoma culture supernatants were screened for anti-EHEC O157:H7 specific antibody with indirect-ELISA. Three strain McAbs were established after the positive clones were subcloned three times by limiting-dilution, which were named as 1E7,7D11 and 3A11, respectively.2. Identification of McAbThe characterizations of the two hybridoma cell lines were preliminarily identified. The ELISA titers of the 1E7 supernatants was 1:2 560 , 7D11 was1:1 280 and 3A11 was1:1 280 while ELISA titers of the 1E7 ascites was 1:6.4×10~6, 7D11 was 1:1.2×10~7and 3A11 was 1:3.2×10~4.Among the three cell strains, 1E7 belonged to IgG1 subclass,7D11 belonged to IgG1 subclass and 3A11 belonged to IgM immunoglobulin subclass.The chromosome number of the three hybridoma cell lines was calculated that average number of the 1E7is 104,7D11 was 102 and 3A11 was 98. Antigen dots and specific tests of 1E7 and 7D11 were analysed. The two McAbs were against different epitopes of EHEC O157:H7. Cross reactivity of the two McAbs with other antigens were not observed(Mycobacterium tuberculosis,Mycobaterium bovis ,Salmonella,StapHylococcus aureus).3. Purification of McAbThe purified protein of McAbs by SDS-PAGE showed that the molecular weights of the heavy chain and light chain of 1E7 was about 45KD和20KD ,and that of 7D11 was about 45KD and 18KD.The affinity of the purifyied products were tested between1E7 and 7D11, and the affinity constant was 1.4×10~6 and 2.8×10~6,respectively.