检测猪睾丸细胞蛋白残留量的间接ELISA的建立  

Establishment and application of an ELISA for determining host cell proteins of swine testis in vaccine

作　　者：暴艳敏[1] 樊得英 冯玉萍[1] 马桂兰 王家敏[1] 马忠仁[1] 李倬[1] 乔自林[1] 

机构地区：[1]西北民族大学甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [2]兰州民海生物工程有限公司,甘肃兰州730010 [3]兰州百灵生物技术有限公司,甘肃兰州730010

出　　处：《中国兽医科学》2015年第10期1091-1096,共6页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"项目(IRT13091);国家民委创新团队计划项目([2013]231);国家自然科学基金资助项目(31460665);甘肃省农业生物技术专项(GNSW-2014-24);西北民族大学中央高校基本科研业务费专项(31920150029);新疆天康畜牧生物技术股份有限公司校企合作项目

摘　　要：为建立猪睾丸细胞疫苗生物制品中宿主细胞蛋白残留量的ELISA检测方法,制备了猪睾丸细胞多克隆抗体,优化了间接ELISA试验条件,建立了ELISA检测方法。Western-blot结果显示,制备的猪睾丸细胞蛋白多克隆抗体具有良好的特异性,抗体经过纯化后效价达1∶10 000。猪睾丸细胞蛋白的质量浓度为25-400ng/mL时线性关系良好。结果表明,用猪睾丸细胞蛋白建立的间接ELISA定量检测方法具有良好的线性关系、特异性和重复性。To establish an ELISA for detection of host cell proteins（HCP）of porcine testis in biologicals,the soluble proteins were prepared from swine testical（ST）cells,and then were injected into rabbits to produce high titers of polyclonal antisera.The titers and specificity of rabbit sera were determined by ELISA and Western-blot,respectively.Using the purified polyclonal antibodies from rabbits,an ELISA was established for determining host cell proteins of swine testis in biologicals.In result,the titers were more than 1∶10 000 and the polyclonal antibodies against ST HCP prepared had a good specificity and reactivity.The polyclonal antibodies have no cross-reactions with DMEM,MEM,newborn bovine serum,fetal bovine serum,and horse serum,respectively.The ELISA method developed had good specificity,sensitivity,and reproducibility and the detection limit was 25 ng per milliliter.The linear range of the standard curve is between 25 to 400ng per milliliter and the correlation coefficient was 0.999.In conclusion,aquantitative ELISA was established for detecting swine testical cell proteins,which provides a reliable method for detection of the host cell proteins in biological products.

关 键 词：酶联免疫吸附试验 猪睾丸细胞 宿主细胞蛋白 

分 类 号：S852.43[农业科学—基础兽医学]