腹水型单克隆抗体纯化方法的研究

Study on purification methods of monoclonal antibody from mouse ascites fluid

目的比较不同纯化方法对腹水型抗体的纯化效果,探索出最适合实验室抗体生产的纯化方法。方法制备的IgG1单克隆抗体腹水(4-D10)分别利用两步硫酸铵沉淀法(AS法)、辛酸-硫酸铵沉淀法(CA-AS法)、辛酸-硫酸铵沉淀法联合DEAE阴离子交换层析法与辛酸-硫酸铵沉淀法联合蛋白G亲和层析法纯化处理后,经蛋白纯度、回收率、抗体免疫活性鉴定分析后,比较不同的纯化方法对腹水型抗体的纯化效果。结果①AS法的蛋白纯度为25%,低于CA-AS法(44%);AS法回收率为63%,高于CA-AS法(41.8%);比较抗体效价表明,AS法(1.024×106)与CA-AS法(1.024×106)两者相当。②粗提后纯化效果表明,蛋白G亲和层析法回收率明显降低,DEAE纯品虽纯度略低,但回收率较高,且效价(5.12×105)高于蛋白G亲和层析法(1.28×105)。结论 CA-AS联合DEAE离子交换法是适合实验室生产单抗的低成本、纯化效果和回收效果较佳的方法。

Objective To compare different methods for purification of antibodies from mouse ascites fluid in order to explore the most suitable laboratory method for purifying antibody. Methods Ammonium sulfate precipitation (AS) and caprylic acid-ammonium sulfate precipitation (CA-AS) methods were used for purification of crude IgG1 antibodies, respectively. The crude samples were further purified by protein G chromatography and DEAE ion exchange chromatography, respectively. Results The purity of antibody purified by AS method was 25%, lower than that of 44%purified by CA-AS method. The antibody recovery by AS method was 63%, higher than that of the CA-AS method 41.8%. The antibody titers obtained by AS method and CA-AS method were almost the same, 1.024 × 106. Further antibody purification by protein G chromatography and DEAE chromatography were performed. The recovery of antibody by protein G chromatography was lower than that by DEAE chromatography. Conclusion CA-AS combined with DEAE chromatography method is the most suitable method for the small scale purification of monoclonal antibody from mouse ascites fluid due to its better recovery, higher purity and lower cost.