单增李斯特菌单克隆抗体制备及免疫磁珠分离方法的建立  

Preparation of monoclonal antibody against Listeria monocytogenes and establishment of an immuno-magnetic bead isolation method

作　　者：叶方钰 谢雨芮 王权[2] 刘爽 张海洋 韩先干[2] 蒋蔚[2] YE Fangyu;XIE Yurui;WANG Quan;LIU Shuang;ZHANG Haiyang;HAN Xiangan;JIANG Wei(College of Animal Science and Technology,Tarim University,Alar 843300,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]塔里木大学动物科学与技术学院,新疆阿拉尔843300 [2]中国农业科学院上海兽医研究所,上海200241 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2023年第9期93-99,共7页Animal Husbandry & Veterinary Medicine

基　　金：上海市科学技术委员会科研计划项目(21N31901000);兵团财政科技计划项目(2020AB025-03);中央级公益性科研院所基本科研业务费专项资金项目(2020JB07)。

摘　　要：免疫磁珠技术操作简单、快速高效,目前已广泛应用于细菌的快速分离纯化等领域,本研究旨在建立一种捕获率高、特异性好的单增李斯特菌免疫磁珠分离方法。首先通过杂交瘤技术制备出8株抗单增李斯特菌单克隆抗体,效价均达到1∶1024000,且特异性较好。利用效价最高的腹水6-E6制备单增李斯特菌免疫磁珠,并对单增李斯特菌免疫磁珠最适条件进行优化,结果选择MEST(7.0)为偶联缓冲液、磁珠直径为750 nm、免疫磁珠添加量为0.4 mg、捕获时间45 min、单增李斯特菌含量在1×10^(4)CFU/mL时,免疫磁珠捕获率最高可达76.29%。在模拟牛奶样品中免疫磁珠特异性捕获率可达到67.71%。该分离方法可快速且特异地分离单增李斯特菌,操作简便,为快速分离食品中的单增李斯特菌并实现快速检测提供了技术支持。The immuno-magnetic bead technology has been widely used in rapid separation and purification of bacteria due to its simple operation,efficiency and other characteristics.The aim of this study was to establish an immuno-magnetic bead separation method for Listeria monocytogenes with high capture rate and good specificity.Eight strains of monoclonal antibodies against Listeria monocytogenes were prepared by the hybridoma technique,with a titer of 1∶1024000 and good specificity.Immuno-magnetic beads of Listeria monocytogenes were prepared using monoclonal antibodies.The optimal conditions for Listeria monocytogenes immuno-magnetic beads were optimized as follows:MEST(7.0)was selected as the coupling buffer,the diameter of the beads was 750 nm,the amount of immuno-magnetic beads was 0.4 mg,the capture time was 45 min,and the content of Listeria monocytogenes was 1×10^(4)CFU/mL.The capture rate of the immuno-magnetic beads reached 76.29%.The specific capture rate of the immuno-magnetic beads in the simulated milk samples was 67.71%.In summary,the present method might be used to isolate Listeria monocytogenes quickly and specifically,which provided technical support for rapid isolation of Listeria monocytogenes in food and rapid detection of the bacterium.

关 键 词：单增李斯特菌 单克隆抗体 免疫磁珠 捕获率 

分 类 号：S852.612[农业科学—基础兽医学]