草甘膦人工抗原的制备及兔源多抗的ELISA鉴定  

Preparation of Artificial Antigen and ELISA Identification of Rabbit Polyclonal Antiserum for the Detection of Glyphosate

作　　者：王晶[1,2] 王耀[2] 王方雨[1] 邓瑞广[1] 胡骁飞[1] 余秋颖[1] 郝俊芳[1] 邢云瑞 侯玉泽[2] WANG Jing;WANG Yao;WANG Fangyu;DENG Ruiguang;HU Xiaofei;YU Qiuying;HAO Junfang;XING Yunrui;HOU Yuze(Henan Key Laboratory of Animal Immunology, Henan Academy of Agriculture Sciences, Zhengzhou 450002, China;Henan Engineering Laboratory of Livestock Disease Diagnosing and Food Safety Testing, College of Food and Bioengineering,Henan University of Science and Technology, Luoyang 471023, China)

机构地区：[1]河南省农业科学院河南省动物免疫学重点实验室,河南郑州450002 [2]河南科技大学食品与生物工程学院,畜禽疫病诊断与食品安全检测河南省工程实验室,河南洛阳471023

出　　处：《食品科学》2017年第4期65-69,共5页Food Science

基　　金：“十二五”国家科技支撑计划项目(2014BAD13B05);河南科技大学博士科研启动基金项目(13480062);河南省基础与前沿项目(142300413222)

摘　　要：采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,EDC)法和戊二醛(glutaraldehyde,GA)法,合成了草甘膦(N-(phosphonomethyl)glycine,PMG)人工抗原,通过免疫制备高敏感性、特异性的兔PMG多克隆抗血清并进行鉴定。将PMG分别与牛血清白蛋白(bovine serum albumin,BSA)和鸡卵清白蛋白(ovalbumin,OVA)偶联,合成免疫抗原PMG-BSA和包被抗原PMG-OVA。经紫外扫描、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定后,免疫新西兰大白兔,制备多抗。利用间接酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)方法测定多抗效价,间接竞争ELISA方法测定血清的敏感性和特异性。结果表明,免疫的3只兔血清效价均达1∶104以上,2号兔多抗效果最好,半数抑制浓度(IC50)为30.9μg/m L,且具备良好的特异性。本实验成功合成了PMG人工抗原,并制得了敏感性高、特异性好的兔多抗,为PMG的免疫学快速检测方法的提供参考。To obtain polyclonal antiserum with high sensibility and specificity,artificial antigens were synthesized bycoupling glyphosate(PMG)with the carrier proteins bovine serum albumin(BSA)and ovalbumin(OVA)separately by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and glutaraldehyde(GA)methods in this study.As aresult,the immune antigen PMG-BSA and detection antigen PMG-OVA were obtained and identified by UV spectroscopyand SDS-PAGE.New Zealand white rabbits were immunized with PMG-EDC-BSA and PMG-GA-BSA,respectively toobtain polyclonal antisera.The titer of the antisera was determined by indirect ELISA and their sensibility and specificitywere determined by indirect competitive ELISA.The results showed that the titer of antiserum of all three immunizedrabbits was higher than1:104.The antiserum of rabbit2exhibited the highest sensibility with an IC50of30.9μg/mL andgood specificity.The successful synthesis of artificial antigens and the obtainment of PMG polyclonal antiserum with highsensibility and specificity may lay the foundation for rapid immunological detection of PMG.

关 键 词：草甘膦 人工抗原 多抗 酶联免疫吸附检测 

分 类 号：S482.4[农业科学—农药学]