Abstract
To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.
Highlights
E. coli O157:H7, a species of enterohemorrhagic E. coli, is the bacterium that causes diarrhea
The Cy-mediated S.I. strategy was used to suppress the immune system of mice to the highly reactive immunodeterminants present on E. coli O157:H19 that was inoculated as the initial immunogen
Sera from control mouse group exhibited a high antibody titer with the E. coli O157:H19, while the sera from mice treated with Cy showed negligible or no detectable reactivity on the E. coli O157:H19 (Fig. 1A)
Summary
E. coli O157:H7, a species of enterohemorrhagic E. coli, is the bacterium that causes diarrhea. In 1996, the most serious E. coli O157:H7 infection in the world occurred in Japan and resulted in 10 deaths and more than 9,000 sick people [1]. Up to now, it has become one of the most important pathogens that caused food borne diseases. Immunological diagnostic methods, which utilize specific antibody, were under consideration due to their simple and rapid protocols. Their efficacies mainly depend on the quality of the specific monoclonal antibodies (mAbs). It is difficult to obtain a high specific antibody against E. coli O157:H7
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