Development Of Immunomagnetic Separation Coupled Fluorescent Microsphere Immunochromatographic Assay And Its Application For Detection Of Escherichia Coli O157:H7

Escherichia coli O157:H7is a typical enterohemorrhagic strain that could cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. E. coli O157:H7has caused outbreaks of diarrhea and associated diseases worldwide. Therefore, it is important to develop on-site detection methods with high sensitivity and specificity for supervision and monitor of this pathogen. Immunochromatographic assay (ICA) with the advantages of rapid, easy operating and high sensitivity is appropriate for primary screening of E. coli O157:H7.Label selection is critical for ICA, among various available labels, colloidal gold is most used. In our previous work, two kinds of labels, fluorescent microspheres (FM) and colloidal gold (CG), were compared for conjugation efficiency when coupled with anti-E.coli O157:H7monoclonal antibodies. Sandwhich-based FM immunochromatographic test strip (FM-ICTS) and CG-ICTS were developed for detection of E. coli O157:H7. This is the first time detailed information of FM/GC-antibody conjugation and their ICTS behavior was investigated. Results showed that the conjugation efficiency of mAbs with FM decreased as increasing of mAbs addition whereas the number of mAb presented on FM surface was increased. When mAb to FM ratio reached100μg/mg, sensitivity of the ICTS reached a plateau. However, conjugation efficiency of mAbs with CG was irregular. When the ratio of mAbs to CG reached10μg/mL, sensitivity of the ICTS didn’t improve. Results showed that the sensitivity of the FM-ICTS was9.6x103CFU/mL, which was five times higher than the CG-ICTS. Only0.1μg mAbs was needed for each FM-ICTS assay while0.4μg for each CG-ICTS assay. Thus, in regarding to the sensitivity and consumption of antibodies, FMs was better than CG as label material for detection of E. coli O157:H7. Moreover, an ’HOOK’ effect can be observed when the concentration of E. coli O157:H7was higher than106CFU/mL, and signal from the test line was decreased for both FM-ICTS and CG-ICTS.Immunomagnetic separation (IMS) technology has been wildly used in detection of pathogenic bacteria. IMS was coupled with FM-ICTS in the present research and been successfully applied for detection of E. coli O157:H7in raw milk. A1mL broth containing E. coli O157:H7was enriched to100μL by IMS, and then applied with FM-ICTS for detection. The whole process of enrichment time could be shortened for up to60min. When0.5CFU/g and2.0CFU/g of E. coli O157:H7were inoculated into raw milk, positive signals could be detected by FM-ICTS after11h and9h preculture, whereas for only9h and7h when combined with IMS. Results suggested that IMS can improve detection specificity and sensitivity, and also eliminate the influences from raw milk matrix.