Rapid Gold Immunochromatography Assays For Detection Of E.Coli O157 And Shiga Toxin

Shiga-producing Escherichia coli, especially serotype O157, have been associated with hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura. Stx(sShiga toxin, Stxs) are the major pathogenicity factors of E.coli O157 which have been implicated in disease. The members of the Stx family of E.coli consist of Stx1, Stx2 and variants of Stx2, however, Stx2 is mostly greater than Stx1 and both of them which secreted by the same E.coli O157 in virulence. It's essential to develop a method to detect directly Stx2. Stx2 has a low pathogenicity dose, and secreted in the culture solution, therefore, detection for Stx2 associated with E.coli O157 was helpful to identify the high pathogenic E.coli O157 isolates. In this study, we had developed sandwich GICA (Gold Immunochromatography Assay) of E.coli O157 and Stx2 to diagnosis the high pathogenic E.coli O157 and Shiga toxin, then we would take necesary steps to control and cure the desease infected by high pathogenic E.coli O157 in outbreak situations.The recombinant expression vector designated as pGEX-Stx2B constructed by the lab was transformed into E.coli BL21 compotent cells, then the positive bacterium colony containing recombinant vector pGEX-Stx2B identified by PCR and digestion of the endonucleases EcoRⅠand XhoⅠhighly expressed fusion protein Stx2B-GST induced by IPTG. It was identified by SDS-PAGE, and was purified by GST-Sepharose Resin. The antisera were prepared respectively by immunizing rabbits (New Zealand White) with purified fusion protein Stx2B and autoclaved E.coli O157 ATCC 43889.The antisera to Stx2B and E.coli O157 were purified by differential (NH4)2SO4-precipitated, caprylic acid-(NH4)2SO4 precipitation, and protein G chromatography respectively. Among the three methods, it showed that the purified IgG against Stx2B by the protein G chromatography have a better effects and easier to prepare a stable complex of rabbit polyclonal antibody against Stx2B conjugated colloidal gold beads. Otherwise, the comparison showed caprylic acid-(NH4)2SO4 precipitation to purify antisera to E.coli O157 was better than the other two methods.According specific lateral-flow and colloidal gold tracing function associated with immunology principle, we established and optimized the sandwich GICA to detect E.coli O157 and Stx2. We optimized reaction system for labelling-antibody in which the optimal amount of the purified anti-Stx2B IgG conjugated with colloidal gold beads was 60μg/mL, the purified antibody for E.coli O157 was 57μg/mL, the optimal pH was 8.2, the size of colloidal gold partical was 20nm, the optimization of stabilizing agent was BSA, the optimization buffer was boracic acid buffer with pH 8.2, the optimization of preserving fluid and eluant was boracic acid buffer with pH 8.2 including 5mM NaCl-1%BSA, confining liquid for NC membrane was 0.01% PBST with 3%BSA, the amount of polyclonal antibody against E.coli O157 and Stx2 conjugated with colloidal gold beads for conjugate pad was 3μg respectively, the amount of anti-Stx monoclonal antibody for test line was 0.1μg and 1μg for E.coli O157, the amount of goat anti rabbit IgG for control line of both GICA were 1μg.A total of 58 isolates identified were assayed in GICA for E.coli O157. The coincidence of GICA with sera agglutination test was 87.5%, the reaction time was 3～5 min, the detection limit for ATCC E.coli O157 43889 was 105CFU /mL, the average CV of positive detection rate in one batch was 2.1%, the average CV of positive detection rate in different batch was 6.9%. The same 58 isolates were assayed in GICA for Stx2. The coincidence of GICA with vero cytotoxicity assays was 50%, the reaction time was 5～10min, the average CV of positive detection rate in one batch was 2.6%, the average CV of positive detection rate in different batch was 28.85%. GICA for E.coli O157 in repeatability and specificity is better to be a potential commercial kit. However, more works should be done to optimize the antibody titer and sensitivity aspects of GICA for Stx2 to advance positive detection rate and repeatability.