【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。更多还原

【Abstract】 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.更多还原