Study On Enrichment And Rapid Detection Of Escherichia Coli O157:H7

O157:H7is an important serotype of Enterohemorrhagic Escherichia coli (EHEC), it causes several disease syndromes, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Most of the outbreaks related to the consumption of contaminated foods and water. So it is extremely important to found a rapid and sensitive method to detect E.coli O157:H7in food. In this study, a novel rapid method by combining immunomagnetic separation (IMS) and gold immunochromatography assay(GICA) was developed for the detection of Escherichia coli O157:H7.Two polyclonal antibodies (PAb) were produced by immunizing rabbit with the whole cells of E.coli O157:H7that were fixed by0.3%formaldehyde. Spleen cells collected from Blab/c mice which immunized with the crude flagelllar antigen of E.coli O157:H7was fused with murine SP2/0myeloma cells. Three murine monoclonal antibodies (MAb)(10C5-H3-B6,8C2and8B1-C2-B1) which reactived with E.coli O157:H7were produced and characterized. The serum and ascetic fluid were purified by the method of caprylic acid-ammonium sulfate precipitation. Then, the purity, titer, specificity and surface antigen binding sites of all antibody were studied. The results of indirect ELISA showed that the titer of the two purification polyclonal antibody were1/6.4×105and1/1.28×106, and the titer of purification monoclonal antibodies10C5-H3-B6,8C2and8B1-C2-B1were1/6.4×106,1/1.6×104and1/1.28×105, respectively. The results of Western-blots indicated that MAb10C5-H3-B6reacted with the lipopolysaccharide (LPS) of E.coli O157:H7and MAb8C2reacted with the H7antigen of E. coli O157:H7.The MAb10C5-H3-B6was coated onto carboxylate-modified superparamagnetic particles via using the EDC and sulfo-NHS compounds as "coupling reagents". Then the immunomagnetic beads (IMBs) were characterized by different tools. Key parameters, including the ratio of MAb to magnetic particles, the amount of IMBs, immunoreaction time and magnetic separation time were optimized. The specificity and the capture efficiency (CE) of IMBs in food matrix were evaluated. The CE of immunomagnetic beads which prepared with MAb10C5-H3-B6was99.9%. The results showed that the reasonable technological parameters were:1mg magnetic beads added with50μg MAb,0.05mg IMBs one time,30min for incubation time and3min for magnetic separation. The specificity of IMBs were very high. The CE of IMBs in spiked ground beef and milk samples was94.4%and99.8%, respectively, and the CE of Dynabeads anti-0157in the same samples was72.8%and95.8%.Colloidal gold was prepared by the method of trisodium citric acid deoxidization, the anti-E. coli O157:H7MAb10C5-H3-B6was selected to conjugate with colloidal gold as the detector antibody, then the immunogold was laid on a conjugation pad. Rabbit polyclonal antibody (No2.) was used as the capture antibody at the test line (T) and donkey anti-mouse IgG antibody was used as the capture antibody at the control line (C) of nitrocellulose strip, then the ready-to-use strip was held in a plastic cassette. A combined immunomagnetic separation (IMS) and gold immunochromatographic assay (GICA) method for a sensitive and rapid detection of E.coli O157:H7was established. A quantitative lateral flow immunoassay for the detection of E.coli O157:H7was developed. The calibration curve was obtained by plotting the ratio between the intensity of the test line (T) and the control line (C) against the logarithmic according to the concentration of E.coli O157:H7. The sensitivity of the combined IMS-GICA methodology was up to7.6×103CFU/mL, demonstrates a10-fold improvement in sensitivity relativing to samples that were applied directly on the GICA without the IMS concentrating step. After20min of incubation at room temperature, the ratio between the intensity of T and C lines (T/C) was tend to be constant. The coefficient of variation (CV) for the strips to detect different concentration of E. coli O157:H7was7.4-10.4%(n=18), the change in T/C was good linearly correlated (R2=0.987) with the logarithmic value of E. coli O157:H7concentration.