5个种属血白蛋白多克隆抗体的制备及其效果评价  

Preparation and efficacy evaluation of polyclonal antibodies against serum albumin of five species

作　　者：张建敏 周长明 李潇 胡宇驰 王铁松 赵璇 ZHANG Jianmin;ZHOU Changming;LI Xiao;HU Yuchi;WANG Tiesong;ZHAO Xuan(Beijing Institute for Drug Control,NMPA Key Laboratory for Research and Evaluation of Generic Drugs,NMPA Key Laboratory of Safety Research and Evaluation of Innovative Drug,Beijing 102206,China)

机构地区：[1]北京市药品检验研究院国家药品监督管理局仿制药研究与评价重点实验室、国家药品监督管理局创新药安全研究与评价重点实验室,北京102206

出　　处：《中国生物制品学杂志》2024年第2期179-182,共4页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2017zx09101001-007);国家药典委员会药品标准制修订研究课题(2018Y007)。

摘　　要：目的 制备人、牛、羊、猪、马血白蛋白多克隆抗体,并评价其用于鉴定人血白蛋白(human serum albumin,HSA)的效果。方法 采用生物信息学技术和人工合成多肽法分别制备人、牛、羊、猪、马血白蛋白特异性多肽,高效液相色谱法鉴定多肽纯度后,再分别与钥孔血蓝蛋白(keyhole limpet hemocyanin,KLH)进行偶联,制备多肽抗原。将5个种属多肽抗原经背部皮下多点免疫雄性新西兰大白兔,2只/种,获得的抗血清经Protein A亲和层析柱纯化,即为多克隆抗体。采用ELISA及Western blot法分别测定多克隆抗体效价及特异性,并使用制备的5个种属血白蛋白多克隆抗体鉴定HSA制品。结果 人工合成的人、牛、羊、猪、马血白蛋白多肽纯度达91%以上,获得相应的多克隆抗体效价均为1∶160 000,可与相应抗原发生特异性结合,且可有效鉴别HSA制品。结论 制备的人、牛、羊、猪、马血白蛋白多克隆抗体具有良好的特异性,且制备过程简单快速,适合大量生产,为HSA快速鉴别试剂盒的研发奠定了基础。Objective To prepare polyclonal antibodies against the serum albumin of human,cattle,sheep,pig and horse,and evaluate their efficacy in the identification of human serum albumin(HSA).Methods The specific polypeptides of human,cattle,sheep,pig and horse serum albumin were prepared by bioinformatics and polypeptide synthesis method,which were coupled with keyhole limpet hemocyanin(KLH)to prepare the peptide antigen after the purity was identified by high performance liquid chromatography(HPLC).Male New Zealand white rabbits were immunized with polypeptide antigens of five species subcutaneously,with 2 for each kind of antigen.The antiserum was then obtained and purified by Protein A affinity chromatography to prepare the polyclonal antibody.The titers and specificity of the polyclonal antibodies were determined by ELISA and Western blot respectively,and the prepared five species of serum albumin were used to identify HSA products.Results The synthetic peptides of human,cattle,sheep,pig and horse serum albumin had a purity of over 91%,and the corresponding polyclonal antibodies all had the titer of 1∶160000,which showed specific binding with the corresponding antigens and effectively identified the HSA products.Conclusion The polyclonal antibodies of human cattle,sheep,pig and horse serum albumin prepared in this study have good specificity and the preparation process is simple and rapid,suitable for the mass production,which lays a foundation of the development of HSA rapid identification kit.

关 键 词：人血白蛋白 多肽 多克隆抗体 免疫双扩散法 

分 类 号：R392-33[医药卫生—免疫学]