T-2毒素单抗制备及免疫学检测方法的建立  

Preparation and preliminary establishment of monoclonal antibody against T-2 toxin

作　　者：郉云瑞 孙亚宁 姚静静 王英华 王方雨 闫文朝 胡骁飞 XING Yun-rui;SUN Ya-ning;YAO Jing-jing;WANG Ying-hua;WANG Fang-yu;YAN Wen-chao;HU Xiao-fei(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang,Henan 471003,China;Key Laboratory for Anitaal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Henan Province Center for Animal Disease Control and Prevention,Zhengzhou 450008,China)

机构地区：[1]河南科技大学动物科技学院 [2]河南省农业科学院动物免疫学重点实验室 [3]河南省动物疫病预防控制中心

出　　处：《中国兽医学报》2018年第7期1338-1345,共8页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2014BAD15B05)

摘　　要：拟制备灵敏度高、特异性强的抗T-2毒素单克隆抗体，并初步建立T-2毒素阻断ELISA检测方法。采用N，N，-羰基二咪唑（CDI）法合成免疫原T-2-BSA和包被原T-2-OVA，经小鼠免疫、细胞融合、利用ELISA筛选技术筛选出分泌抗T-2毒素单克隆抗体的杂交瘤细胞株，采用腹腔注射细胞株制备腹水，并进行免疫学特性鉴定，初步建立T2毒素阻断ELISA检测方法。结果显示，筛选出能稳定分泌抗T-2毒素单克隆抗体的杂交瘤细胞株l株命名为4F7，经鉴定该细胞株分泌的单克隆抗体（iTIAb）效价高达1．024×10^6，亚型为IgG1型，亲和力常数为1．49×10^14mol／L，IC50为2．19μg／L，检测限LOD为0．07μg／L，与其他霉菌毒素及载体蛋白BSA和OVA交叉反应率均小于0.01％。样品回收率为83．8％-94．3％，变异系数小于15％。本试验制备出了灵敏度高、特异性强、亲和力高的T-2毒素单克隆抗体，并建立了T-2毒素的阻断ELISA快速检测方法，为T-2毒素检测提供了技术支撑。This study aimed to prepare monoclonal antibodies against T-2 toxin with high sensitivity and specificity,and preliminary establish the indirect competitive ELISA method for T-2 toxin de- tection. Immunogen T-2-BSA and coating antigen T-2-OVA were synthesized by using N, N-car- bonyldiimidazole method（CDI）, then BALB/c mice were immunized with T-2-BSA. The hybrido- ma cell lines secreting monoclonal antibodies against T-2 toxin were screened out by cell fusion technique and ELISA method. Ascites were prepared by in vivo method, and its immunological characteristics were evaluated. Finally, based on the above results, the indirect competitive ELISA method for T-2 toxin was preliminary established. One hybridoma cell line of 4F7 which stably se- creted monoclonal antibodies（mAb） against T-2 toxin was screened out. The titer of the mAb rea- ches up to 1 ： 1. 024 × 10^6 ,the isotype is IgG1, the affinity constant is 1.49×10^11 mol/L,IC50 is 2.19 μg/L,LOD is 0.07 μg/L,and all the cross reaction rates of mAb with other myeotoxins,car rier protein BSA and OVA were lower than 0.01%. The recoveries were 83.8%-94.3% and the coefficient of variation was less than 15%. In this study,based on the monoclonal antibody against T-2 toxin with high sensitivity,high specificity and high affinity,the indirect competitive ELISA method for T-2 toxin was established,providing the technical support for the rapid detection of T-2 toxin.

关 键 词：T-2毒素 人工抗原 单克隆抗体 阻断ELISA 

分 类 号：S852.44[农业科学—基础兽医学]