| Objective: Escherichia coli O157:H7 is one of the important pathogens that has become a threat to human health. It makes the sources of infection control, monitoring and tracking becomes a problem because of its highly pathogenic, low-dose infection and long incubation period. Therefore, It is necessary to develop a rapid, simple,sensitive and specific detection methods to ensure food safety.Methods: Colloidal gold was prepared by thisodium citrate method. The gold nanoparticles were detected by UV spectrophotometer and transmission electron microscopy. Then, It was labeled to the Escherichia coli 0157:H7 monoclonal antibody(McAb to E.coli O157:H7).The optimal conditions for preparing the collidal gold conjugates with McAb to E.coli O157:H7 were compared.Finally,a gold immunochromatographic assay(GICA)for detection E.coli O157:H7 was developed with that McAb to E.coli O157:H7 was conjugated with collidal gold,which laid on a glass fiber membrane,McAb to E.coli O157:H7 and rabbit anti-mouse IgG antibody were used at the test line (T) and control line (C) respectively. The various factors and conditions of GICA were explored.The optimum coating concentration of antibodies and dilution multiple of the colloidal gold labeled antibody buffer were ascertained.And different concentrations of purified Escherichia coli 0157:H7 bacterial suspension were observed in order to verified the sensitivity, specificity, repeatability and stability of the method.On the basis of gold immuno-chromatographic assay combined with immunomagnetic separation,the rapid immunoassay for the detection of E.coli O157:H7 was established.The magnetic beads coated with sheep anti-mouse IgG binding to the specific McAb to E.coli 0157: H7 were used to captured the E.coli 0157:H7 from the sample,then the bacterial enriched by magnetic beads was separated from the beads and resuspended.The bacterial suspensions were detected using the same GICA strip to evaluate the sensitivity and specificity of this new method. At last,150 simulated food samples including beef,water and milk were used for test with both the established test kit and the traditional gold standard method. Assess the coincidence rate of the detection and evaluate their advantages and disadvantages.Results: Successfully prepared 20nm colloidal gold.The average diameter is 20±3nm,It had good uniformity in shape and had good dispersion as well. The optimal conditions for preparing the colloidal gold conjugates with McAb to E.coli O157:H7 were compared and found that pH 8.0 and McAb protein in 16.8μg/ml could get best results.The optimal concentrations for immunogold which diluted at the rate of 1:1 on the conjugate pad,1mg/ml for T line,and 5mg/ml for C line on the membrane. The clear bands were shown within 5 mins. With colloidal gold immunochromatography test strip for the pure E.coli 0157:H7 bacteria suspension,the detection sensitivity was 105CFU/ml.It exhibited good specificity,stability,and repeatability was also well. When colloidal gold immunochromatography test strip combined immunomagnetic separation technologies, the sensitivity was 103CFU/ml, and the specificity was similar.In stimulated food samples, the test results were correspond to the gold standard method.Conclusion:The E.coli O157:H7 GICA strip was a kind of rapid,sensitive, specific method.In addition, the colloidal gold immunochromatographic assay combining immunomagnetic separation, the sensitivity of detection can be increased by two orders of magnitude,and had good specificity and broad applicability. It had important social significance and economic benefits,applicable to performing the tests at point of care.
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