单核细胞增生李斯特菌特异性单链抗体磁珠的制备与评价

Preparation and Evaluation of Listeria monocytogenes Specified Single-chain Antibody Fragments Immuno-magnetic Beads

目的:制备一种单核细胞增生李斯特菌的特异性单链抗体捕获磁珠(scFv-IMBs),通过用人工污染的方式评价scFv-IMBs的效果。方法:在不同的增菌培养体系中以市售磁珠(Dyna-IMBs)作为对照,通过定性、定量培养技术和PCR检测技术,分析scFv-IMBs在法兰克福香肠中捕获与分离单核细胞增生李斯特菌和英诺克李斯特菌的能力。结果:在BHI培养基中,scFv-IMBs对单核细胞增生李斯特菌的捕获率介于15%~55%之间,优于Dyna-IMBs(0.85%~1.27%),显示出良好的特异性。在MOPS-BLEB培养基中两种磁珠的捕获率在62%~88%,捕获率高于在UVM培养基(<5%)和FB培养基(<53%)中。人工污染样品经scFv-IMBs捕获,单核细胞增生李斯特菌的菌落数在选择性培养基上的比例有显著性提升(P<0.01),可从高浓度英诺克李斯特菌(1∶1000)中有效分离单核细胞增生李斯特菌。磁珠检测方法的灵敏度可达1 CFU/mL。结论:与市售免疫磁珠产品相比,scFv-IMBs具有较好的特异性,可显著降低体系中英诺克李斯特菌的干扰,具有更好的分离能力。

Objective:To preparation a Listeria monocytogenes specified phage-display single-chain antibody fragments immuno-magnetic beads(scFv-IMBs),the effectiveness of scFv-IMBs was evaluated by artificial contamination.Method:the capture efficiency and the ability of scFv-IMBs for separating L.monocytogenes from L.innocua in frankfurters was compared to commercially available anti-Listeria immunomagnetic beads(Dyna-IMBs)using qualitative and quantitative culture method,as well as PCR method,in various enrichment systems.Result:the capture efficiency of scFv-IMBs for L.monocytogenes was 15%-55%,which is better than the efficiency of Dyna-IMBs(0.85%-1.27%),and scFv-IMBs shows good specificity.The capture efficiency of those IMBs in MOPS-BLEB could reach 62%-88%,which was better than IMBs in UVM media(<5%)and FB media(<53%).Moreover,the scFv-IMBs displayed higher specificity to L.monocytogenes(P<0.01)even when the initial cells concentration of L.innocua were 1000 times more than that of L.monocytogenes spiked in commercial frankfurters exudates.And the detection limitation combined with scFv-IMBs could reach 1 CFU/mL.Conclusion:scFv-IMBs have better specificity and separation ability than Dyna-IMBs,which could significantly lower the interference of L.innocua in complex matrix.