脱氧雪腐镰刀菌烯醇单克隆抗体的制备及间接竞争ELISA方法建立  

Preparation of monoclonal antibody against deoxynivalenol and establishment of indirect competitive ELISA

作　　者：孙亚宁[1] 李青梅[1] 杨苏珍[1] 胡骁飞[1] 杨继飞[1] 张颖硕 陈琳琳 邢云瑞 邓瑞广[1] 张改平[1,2] SUN Yaning;LI Qingmei;YANG Suzhen;HU Xiaofei;YANG Jifei;ZHANG Yingshuo;CHEN Linlin;XING Yunrui;DENG Ruiguang;ZHANG Gaiping(Henan Provincial Key Laboratory of Animal Im munology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2021年第4期689-696,共8页Chinese Journal of Veterinary Science

基　　金：中国博士后科学基金资助项目(2018M632778);河南省农业科学院科研发展专项基金资助项目(2019CY04);“十二五”国家科技支撑计划资助项目(2014BAD13B05);河南省农业科学院科技创新创意基金资助项目(2020CX20)。

摘　　要：为了快速准确地检测脱氧雪腐镰刀菌烯醇(DON)污染、控制其危害,本研究制备了高灵敏度的DON单克隆抗体,并建立了间接竞争酶联免疫吸附(ELISA)快速检测方法。采用N,N′-羰基二咪唑(N,N′-Carbonyldiimidazole,CDI)一步法将DON与载体蛋白BSA及OVA偶联制备人工抗原。用DON-BSA免疫小鼠,并根据免疫效果在常规免疫方案中增加腹腔免疫进行改进,利用细胞融合技术,获得能稳定分泌DON单克隆抗体的杂交瘤细胞株1株2A3-E11。经鉴定该细胞株分泌的DON抗体为IgG1型,效价达1∶1.024×10^(6),IC_(50)为21.5079μg/L,亲和力可达9.064×10^(8) mol/L;与15-AcDON有233.7%的交叉反应,与其他结构类似物及常见真菌毒素未见明显交叉反应。基于该抗体建立的间接竞争ELISA检测方法检测范围为4.6968~98.4900μg/L,小麦样品中的加标回收率为90.509%~116.016%,变异系数为3.081%~7.928%。研制的间接竞争ELISA检测方法灵敏度高、结果可靠,可进一步应用于小麦等谷物样品中DON污染的检测,为DON的免疫学检测技术研发奠定基础。The objective of the experiment is to prepare high sensitivity monoclonal antibody against deoxynivalenol(DON)and establishment of indirect competitive ELISA for preventing the DON harm.DON was coupled with bovine serum albumin(BSA)by N,N′-Carbonyldiimidazole(CDI)method.The hybridoma cell clones 2A3-E11secreting stable monoclonal antibodies against DON were obtained,the anti-DON monoclonal antibody was generated and characterized,its subtype was IgG1.The titers and IC_(50)were 1∶1.024×10^(6)and 21.5079μg/L,respectively.The affinity constant(Ka)was 9.064×10^(8) mol/L.The cross-reactivity(CR)of the anti-DON monoclonal antibody with 15-AcDON was 233.7%and no obvious cross reaction to other structural analogue and mycotoxins.The detection range of the indirect competitive ELISA was 4.6968-98.4900μg/L.The recoveries of DON spiked in wheat were 90.509%-116.016%,the coefficient variation was 3.081%-7.928%.The indirect competitive ELISA developed in this study is highly sensitive and reliable,which can be used to DON detection in wheat and other grains.This study lays a foundation for DON immunological detection technology.

关 键 词：脱氧雪腐镰刀菌烯醇 免疫程序 单克隆抗体 酶联免疫吸附测定 

分 类 号：S859.87[农业科学—临床兽医学]