Combination Of Immunomagnetic Beads Separation And Fluorescence Immunochromatographic Assay For Detecting Escherichia Coli O157:H7

Escherichia coli O157:H7is one of the main pathogens of Escherichia coli, it was firstdiscovered in1982. Infected with E. coli O157will cause infant diarrhea, hemorrhagic colitis,hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and death, etc.10%ofthe cases will lead to2%~10%mortality rate. E. coli O157has caught public’s attentiondueto its hightoxicity. It’s meaningful to establisha rapid, simple, high sensitivity and specificitydetection method for monitoring E. coli O157: H7.Combination of immunomagnetic beads and fluorescent latex immunochromatography issimple, quick and high sensitivity, which can realize quantitative or semi-quantitativeoperation, without enrichment and any complex instruments, the whole process can be donewithin15minutes and is very suitable for field testing.This research has developed a fluorescent latex immunochromatographic stripsuccessfully. The fluorescent latex is coated with anti-O157:H7monoclonal antibodies. The Tline is coated with anti-O157:H7monoclonal antibodies and C line is coated with sheepanti-rabbit IgG. This experiment screened suitable antibody, assembled test card, optimizedand tested the sensitivity, accuracy, precision and specificity,etc.This research also discussedthe best condition for the synthesis of immunomagnetic beads, best working concentration ofIMB and capture efficiency. At last, simulated food samples includingbeef,waterand Vitasoywereused fortestwithboth theestablishedtestkit.The best dilutionratio of T, C line is100x,1000x, separately. T McAb in0.1mg/mL andC McAb in0.5mg/mL get best result. The ratio of bacteria and Immunofluorescence latexsolutionis1:1. Best drying time of the strip is3days, fluorescencesignal is stable in15min.The strip has detection sensitivity of105CFU/mL, good specificity, stability andrepeatability. E. coli O157:H7can be collected over90%by immunomagnetic beads. Theoptimization for IMB is: pH6.0MES buffer when coupling antibody to MB; Mass ration ofMB and antibody is3.3; Best capturing time is30min. When fluorescent lateximmunochromatography combined with immunomagnetic technology, all positive samplescan be detected after immunomagnetic beads enrichment, and the limit of detection can down to103CFU/mL.Ithadimportant socialsignificanceand economic benefits,applicabletoperformingthetestsatpoint of care.