Development Of A Rapid Colloid Gold Immunochromatographic Metod For Detection Of Escherichia Coli O157:H7

Escherichia coli (E. coli) O157:H7 is an important foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome in human. Human infections with E. coli O157:H7 have usually been found to follow the consumption of contaminated, improperly prepared beef products, dairy foods, and vegetables. In addition, direct animal-to-person and person-to-person contact can result in E. coli O157:H7 transmission. It has seriously affected the health of human because its fast invasion and high mortality when infections. Hence, it has important practical significance of developing a rapid method for diagnosis E. coli O157:H7. In this study, a colloid gold immunochromatographic method which is rapid, high specificity and suitable for a great number of samples and on site detection was established for detection of E. coli O157:H7.Japanese white rabbits were immunized with the whole cell and cell debris of E. coli O157:H7 to prepare polyclonal antibody. The titer of antibody was 106. Caprylic acid ammonium sulphate precipitation and magnetic beads adsorption were used to purify polyclonal antibodies (pAb). Then SDS-PAGE was used for proteins analysis while Dot blot hybridization was used for analysis the specificity of pAb. The SDS-PAGE results showed that antibodies purified by caprylic acid ammonium sulfate precipitation still had many other proteins; however, the magnetic beads adsorption could effectively remove the other proteins and get pure immunoglobulin. The antiserum after purified reacted with other bacteria.In this study, commercial anti-E. coli O157:H7 monoclonal antibody was labeled with colloidal gold and anti-E. coli O157:H7 polyclonal antibody and donkey anti-mouse IgG were dispensed on the nitrocellulose membrane as the test line and control line respectively. The strips parameters were as follows:The reaction system pH was 8.0, anti-E. coli O157:H7 monoclonal antibody labeling with gold particles was 25μg/mL, the gold-antibody conjugate was centrifuged at 4000 rpm/min and blocked with PEG 20000, lmg/mL polyclonal antibody and 1.5 mg/mL donkey anti-mouse IgG were dispensed for the test line and control line respectively. The senitivity and specificity of the strip were determined using pure cultured bacteria. E. coli O157:H7 could be detected at a minimum of 105 cells/mL. Cross reaction test with 26 bacteria strains showed that only Staphylococcus aureus (CMCC 26003) and Salmonella typhimurium (ATCC 13311) had slight cross reaction, whereas the rest 24 strains had no cross reaction. The preservation test at 37℃indicated that the strips could be stored at room temperature for more than 15 months. The strips showed positive result after inoculating E. coli O157:H7 in samples (beef or lettuce) at 10 to 100 CFU/25 g and incubating for 6 h.In order to increase the specificity and sensitivity of the assay, Balb/c mice were immunized with flagellum proteins isolated from E. coli O157:H7. After fusion and cloning, four hybridoma cells which would stable secret antibodys against E. coli O157:H7 were abtained and prepared ascitic fluid. The matching results showed that 5 pairs of antibodies could be used for strip preparation.