Development Of Colloidal Gold Immunochromatographic Strip For The Detection Of Enterohemorrhagic E. Coli O157:H7

Food-borne pathogenic microorganisms are the primary causes of the food safety and public health events, in food-borne pathogenic microbes, enterohemahagic E.coli O157:H7is one of the important pathogens. The objective of the experiments was to establish specific monoclonal antibodies against0157:H7and develop the colloidal gold immunochromatographic strip for the rapid detection of0157:H7.The paper contains three parts:1. Establishment of the Hybridoma Producing Monoclonal Antibodies against Enterohemorrhagic E.coli O157:H7Seven hybridomas secreting monoclonal antibodies (mAbs) against enterohemahagic E.coli O157:H7(EHEC-O157:H7)were established by fusing SP2/0with spleen cells from BALB/c mice immunized with formaldehyde-killed and ultrasonically-broken E.coli O157:H7, naming2E7,2F5,1G9,1D3,2H7,2G11and1C3. The Ig isotypes of mAbs were IgG2b κ、IgG2a κ、IgM κ、IgM κ、IgG2b κ、IgM κ and IgG2a κ, respectively. The ELISA antibody titers of of1G9and2G11were1.6×103and1×1011in supernatant and ascitic fluid respectively, the others were1×101～2×10in supernatant, and4×102～1×106in the ascitic fluids. Additivity ELISA indicated that1D3and2H7were corresponding to the same antigen epitope, but the other5mAbs were different epitopes.2E7,2H7and1D3were specific anti-E. coli O157:H7mAbs, the other4mAbs could react with some other kinds of bacteria in the specific analysis by indirect ELISA. In the western-blotting, mAbs1C3,1G9,2G11,2E7,1D3and2H7displayed the stained protein band of approximate35kD, but the mAb2F5didn’t show any stained protein band.2Development of a Gold Immunochromatographic Strip for the Detection of E. coli O157:H7The gold particle in colloidal solution was coupled with purified monoclonal antibody 2E7, and then sprayed on the glass fibers. The purified monoclonal antibody2H7and Goat anti-mouse IgG served as 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 sequence. The detection results indicated that the strip was specific to O157:H7and the sensitivity was106CFU/ml, which was equivalent to the sandwich ELISA established by using the same monoclonal antibodies2E7and2H7. The IC strip is more convenient, rapid andsimple for the clinical diagnosis and investigation of E.coli O157:H7infection or/and contamination.3Aplication of the gold immunochromatographic strip for the detection of cattles and mice inoculated with E. coli O157:H7To investigate the aplication of the gold immunochromatographic strip for the detection of animals inoculated with E. coli O157:H7. Cattle and mice were inoculated intragastrically with E. coli O157:H7, and their faeces were detected using the three methods of gold immunochromatographic strip, counting bacterial colony in identification medium and polymerase chain reaction (PCR). E. coli O157:H7in faeces could be detected on day2in the inoculated catties and the peak levels of O157:H7in faeces reached on the forth day and then it dropped down gradually up to day28. In the inoculated mice, O157:H7could be detected in faece at4hours and the peak levels at6hours after inoculation, and then it dropped down gradually up to day15. The results were consistent with the three methods of colloidal gold immunochromatographic strip, counting bacterial colony and PCR for the detection of E.coli O157.H7. However, the gold immunochromatographic strip is convenient and rapid for the clinical diagnosis and field investigation of E.coli O157:H7infection or contamination.