基于电融合方法的牛妊娠相关糖蛋白单克隆抗体制备及其应用  

Preparation and Application of Monoclonal Antibody against bPAG by Electrofusion-based Protocol

作　　者：尹茉莉 聂元旺 周婉婷 张爽 刘磊[1] 冀慧敏 刘方圆 王会岩[1] YIN Mo-li;NIE Yuan-wang;ZHOU Wan-ting;ZHANG Shuang;LIU Lei;JI Hui-min;LIU Fang-yuan;WANG Hui-yan(Jilin Collaborative Innovation Center for Antibody Engineering,Jilin Medical University,Jilin 132013,China;Jilin City Garden Management Center,Jilin 132013,China;Jilin Teyan Biotechnology Co.,Ltd.,Changchun 130112,China)

机构地区：[1]吉林医药学院吉林省抗体工程科技协同创新中心,吉林吉林132013 [2]吉林省吉林市园林管理中心,吉林吉林132013 [3]吉林特研生物技术有限责任公司,吉林长春130112

出　　处：《中国兽医杂志》2022年第12期25-30,共6页Chinese Journal of Veterinary Medicine

基　　金：吉林省科技厅技术攻关项目(20190301088 NY)。

摘　　要：为了制备抗牛妊娠相关糖蛋白(bPAG)单克隆抗体,本试验提纯妊娠母牛子叶bPAG,免疫小鼠后进行细胞电融合,通过ELISA、Western blot方法对抗bPAG单克隆抗体进行筛选。结果显示:获得5株bPAG特异性强、灵敏度高的单克隆抗体,抗体最高效价为1∶256000,亲和力最高可达到3.75×10^(7) L/mol,5株单克隆抗体均能识别天然bPAG蛋白。采用高碘酸钠法对单抗标记辣根过氧化物酶(HRP),将最佳配对抗体用于建立双抗夹心ELISA方法,对64个血清样本进行检测。结果显示:以10H2作为捕获抗体与HRP-5G1配对建立的双抗夹心ELISA方法检测bPAG阳性和阴性血清样本,与爱德士牛早孕检测试剂盒比较符合率达95.2%。结果表明,本试验运用细胞电融合法制备的抗bPAG单克隆抗体可应用于建立双抗夹心ELISA体系,为下一步bPAG检测试剂盒的研制提供技术支持。In order to prepare monoclonal antibodies against bovine pregnancy associated glycoprotein(bPAG),this study immunized mice with purified bPAG of pregnant cow and utilized electrofusion apparatus for cell fusion.The resulting monoclonal antibodies to bPAG protein were screened by ELISA and Western blot.The results showed that five monoclonal antibodies against bPAG with high specificity and sensitivity were obtained,with the highest titer of 1∶256000 and the highest affinity constant of 3.75×10^(7) L/mol.All the five monoclonal antibodies could recognize the natural bPAG protein.Monoclonal antibodies were labeled with horseradish peroxidase(HRP)by sodium periodate method and evaluated in a double antibody-sandwich ELISA using 64 serum samples.The results showed that the constructed double antibody-sandwich ELISA using two optimal paired antibodies10H2 and HRP-5G1 displayed coincidence rate of 95.2%with IDEXX Bovine Pregnancy Test kit,when detecting the positive and negative serum samples of bPAG.Thus,monoclonal antibodies against bPAG were successfully prepared by electrofusion method and applied in double antibody-sandwich ELISA,making future development of bPAG detection kit a distinct possibility.

关 键 词：牛妊娠相关糖蛋白 电融合 单克隆抗体 双抗夹心ELISA 

分 类 号：S823.91[农业科学—畜牧学]