免疫亲和层析法纯化抗Pgp/抗CD3双功能抗体  

Study on purification of anti-CD3/anti-Pgp diabody by the immunoaffinity chromatography

作　　者：王金宏[1] 刘娟妮[1] 高瀛岱[1] 邵晓枫[1] 熊冬生[1] 杨纯正[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《中国免疫学杂志》2008年第4期356-359,共4页Chinese Journal of Immunology

基　　金：天津市科技发展计划项目(No05WFGPGX06900);国家自然科学基金资助项目(No30400558)

摘　　要：目的:制备可以纯化抗Pgp/抗CD3双功能抗体的免疫亲和层析柱。方法:纯化的抗抗CD3ScFv单克隆抗体与预活化的Sepharose4B偶联制成免疫亲和层析柱,采用自制的免疫亲和层析柱纯化由摇瓶发酵获得的抗Pgp/抗CD3双功能抗体,采用间接免疫荧光法测定抗CD3/抗Pgp微型双功能抗体能与Jurkat细胞及K562/A02细胞特异性结合活性。结果:成功地制备了可以纯化抗Pgp/抗CD3双功能抗体的免疫亲和层析柱,采用此柱纯化的抗体与Jurkat细胞及K562/A02细胞特异性结合的活性与带有E-tag纯化标志的抗体基本一致。结论:此介质可以替代价格高昂的E-tag亲和层析介质在纯化Pgp/抗CD3双特异双功能抗体中的应用,同时还可以避免由于E-tag纯化标志而带来的免疫原性问题,此项研究工作为抗Pgp/抗CD3双功能抗体将来在临床应用奠定了基础。Objective：To prepare the immunoaffinity chromatography column which can purify anti-CD3/anti-Pgp diabody.Methods：McAb against anti-CD3 ScFv was coupled with activated sepharose 4B to prepare the immunoaffinity chromatography column for purification of anti-CD3/anti-Pgp diabody.Anti-CD3/anti-Pgp diabody was expressed in E.coli strain 16C9 transformed with plasmid pAYZDCP in shaking flask.The diabody was recovered after immunoafinity chromatography purification and predominantly as a dimer.Anti-CD3/anti-Pgp diabody and anti-CD3/anti-Pgp diabody（E-tag）binding to CD3-positive Jurkat cells and Pgp-positive K562/A02 cells was analyzed by FACS,respectively.Results：The affinities of anti-CD3/anti-Pgp diabody and anti-CD3/anti-Pgp diabody（E-tag）binding to CD3-positive Jurkat cells and Pgp-positive K562/A02 cells were similar.Conclusion：The result of biological activity experiments indicates that the prepared immunoaffinity chromatography column can partly substitute for anti-E tag chromatography column,which may purify ScFv antibody.

关 键 词：抗抗CD3ScFv单克隆抗体 抗Pgp/抗CD3双功能抗体 亲和层析 

分 类 号：R392.11[医药卫生—免疫学]