双抗体夹心ELISA法检测食品中出血性大肠杆菌O15
Establishment of double- antibody sandwich ELISA for detection of EHEC O157 in food
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摘要: 以灭活重组基因工程菌EHEC O157Δler/stx（p BR322::stx1/2B::eae）免疫新西兰大耳白兔获得的兔多抗作为捕获抗体,以针对出血性大肠杆菌O157主要毒力因子的特异性单抗2H9为检测抗体,建立双抗体夹心ELISA方法并绘制标准曲线用于食品中出血性大肠杆菌O157的定量分析。标准曲线线性方程为y=0.3479x-0.4121,R2=0.9862。该ELISA的重复性变异系数小于5%,稳定性良好。应用该方法测得出血性大肠杆菌O157∶H7 EDL933的最小检出限为103cfu/m L。食品样品接种实验表明:牛肉或猪肉样品接种量为0.08 cfu/g,增菌8 h后可以检出阳性反应;蔬菜和牛奶样品接种量为0.12 cfu/g（m L）,增菌10 h后可以检出阳性反应。Abstract: The polyclonal antibody of Enterohemorrhagic Escherichia coli O157( EHEC O157) Ig G was prepared by intravenous injection into New Zealand rabbit with inactivated engineered bacteria E.coli O157 Δler/stx( p BR322: :stx1 /2B: : eae).A double-antibody sandwich ELISA was developed to detect E.coli O157 in food samples,which using polyclonal antibody Ig G as the capture antibody,and monoclonal antibody 2H9 against main virulence factors of EHEC O157 as the detection antibody.Linear equation of standard curve was y = 0.3479x-0.4121,R2= 0.9862,and variation coefficient less than 5%. The detection limit of this sandwich ELISA was 10~3 cfu/m L E.coli O157 in pure culture.Inoculation test of EHEC O157 in food samples indicated that the positive result could be detected when inoculated levels were 0.08 cfu/g with 8 h enrichment in negative beef or pork and 0.12 cfu/g( m L) with 10 h enrichment in negative vegetables and milk.
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