两步串联层析法纯化鼠抗人CD80单克隆抗体4E5  

Two-step Tandem Chromatography Purification of Anti-human CD80 Monoclonal Antibody 4E5 from Mouse Ascites

作　　者：马泓冰[1] 邱玉华[1] 陶然[1] 李文香[1] 徐颖[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,苏州215007

出　　处：《中国生物工程杂志》2006年第8期84-87,共4页China Biotechnology

基　　金：国家自然科学基金重点资助项目(30330540)

摘　　要：采用阴离子交换与凝胶过滤两步串联层析法，纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后，在Tris-HCl缓冲溶液（pH8．0，50mmol／L）条件下，上阴离子交换柱对目的单抗进行捕集，采用0-0．5mol／L NaCl浓度分步洗脱；含目的单抗的洗脱馏分再上凝胶过滤柱纯化，用PB缓冲溶液（pH7．2，20mmol／L）洗脱，获得目的单抗4E5，其生物学活性高、纯度大于95％，抗体总回收率达61％。A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0, 50 mmol/L Tris-HCl and satisfactory results were obtained using a 0 - 0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

关 键 词：阴离子交换层析法 凝胶过滤层析法 单克隆抗体 腹水 纯化 

分 类 号：Q511[生物学—生物化学]