T-2毒素完全抗原制备及免疫学检测方法的建立  

Preparation of T-2 Toxin Complete Antigen and Establishment of Immunological Detection Method

作　　者：郉云瑞 孙亚宁[1] 姚静静[1] 王英华[2] 郝慧芳[1] 柴书军[1] 李俊鹏 胡骁飞[1] XING Yunrui1, SUN Yaning1, YAO Jingjing1, WANG Yinghua2, HAO Huifang1, CHAI Shujun1, LI Junpeng3, HU Xiaofei1(1. Key Laboratory for Animal Immunology, Henan Academy of Agricultiural Sciences, Zhengzhou 450002, China;2. Henan Province Center for Animal Disease Control and Prevention, Zhengzhou 450008, China;3 . Pingdingshan Academy of Agricultiural Sciences, Pingdingslian 467000, Chin)

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]河南省动物疫病预防控制中心,河南郑州450008 [3]平顶山农业科学院,河南平顶山467000

出　　处：《河南农业科学》2018年第4期124-129,共6页Journal of Henan Agricultural Sciences

基　　金：十二五国家科技支撑计划项目(2014BAD15B05);河南省农科院“四优四化”科技支撑行动计划项目

摘　　要：为建立小分子霉菌毒素中T-2毒素残留的检测方法,合成并鉴定T-2毒素完全抗原,通过制备敏感性强的抗T-2毒素多抗血清,建立T-2毒素免疫学检测方法。采用N,N'-羰基二咪唑(CDI)法合成完全抗原T-2-BSA和包被原T-2-OVA,采用聚丙烯酰胺凝胶电泳(SDSPAGE)和动物免疫试验对合成的完全抗原进行鉴定,优化ELISA反应条件,建立T-2毒素间接竞争ELISA检测方法。SDS-PAGE结果显示,完全抗原T-2-BSA合成成功;动物试验结果显示,制备的完全抗原能够刺激小鼠产生针对T-2毒素的特异性抗体;基于该抗体所建立的T-2毒素间接竞争ELISA检测方法的灵敏度(IC50)为8.34 ng/m L,检测限(LOD)为0.048 ng/m L。综上,成功合成了完全抗原T-2-BSA,获得了效价高、敏感性强的抗T-2毒素的鼠源多克隆抗体,并初步建立了T-2毒素免疫学检测方法。In order to detect the residue of T-2 toxin,the complete antigens of T-2 toxin were synthesized and identified,and an immunological detection method of T-2 toxin based on T-2 antiserum with high sensitivity was established.T-2 toxin was conjugated with BSA and OVA using N,N＇-carbonyl imidazole （CDI） method,and complete antigens were identified by SDS-polyacrylamide gel electrophoresis（SDS-PAGE） and animal immune experiment.After optimizing the reaction conditions of ELISA,indirect competitive ELISA method of T-2 toxin was established.SDS-PAGE results showed that the complete antigen was synthesized successfully,meanwhile the results of animal experiments indicated that complete antigen could induce mice to produce specific antibody against T-2 toxin.The sensitivity （IC50） of the indirect competitive ELISA to assay T-2 toxin was 8.34 ng/mL,and the limit of detection （LOD） was 0.048 ng/mL.In conclusion,we successfully synthesized the complete antigen T-2-BSA and obtained the polyclonal antibody against T-2 toxin with high titer and high sensitivity,and developed preliminarily immunological detection method of T-2 toxin.

关 键 词：T-2毒素 完全抗原 多克隆抗血清 ELISA 

分 类 号：S859.84[农业科学—临床兽医学]