title: A rapid subtractive immunization method to prepare discriminatory monoclonal antibodies for food E. coli O157:H7 contamination.
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 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) 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.
authors.affiliation: Department of Environment and Health, Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment and Food Safety, Tianjin, China.
authors.forename: Min
authors.initials: M
authors.lastname: Jin
authors.forename: Jing
authors.initials: J
authors.lastname: Lang
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Shen
authors.forename: Zhao-Li
authors.initials: ZL
authors.lastname: Chen
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Qiu
authors.forename: Xin-Wei
authors.initials: XW
authors.lastname: Wang
authors.forename: Jun-Wen
authors.initials: JW
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D006825:Hybridomas; D007114:Immunization / Q000379:methods*; D007118:Immunoassay / Q000379:methods; D008722:Methods; D051379:Mice; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal