摘要
TNT-1是-种鼠IgG_2a单抗,已用于肿瘤病人的临床试验。本文将介绍从腹水中进一步纯化TNT-1的方法及制备其F(ah′)_2片段的条件。经Protein A亲和柱层析从小鼠腹水中提取出的lgG,再通过阳离子交换树脂柱Mono S,用缓冲液A(20 mmol/L MES,pH6.3)和缓冲液B(lmol/L NaCI),在快速蛋白液相色层(FPLC)系统上进行梯度洗脱分离,可制得纯净的TNT-1抗体,其免疫活性可达80％～85％;经纯化的TNT-1,用胃蛋白酶消化,制备F(ah′)~2片段,其消化条件是:酶的用量为抗体的1/250,pH3.8,温度37℃,时间4～5 /小时。消化后的混合物,用protein A亲和结合法除去完整的IgG,其余的F(ab′)_2和Fab、Fc等片段,在Mono S柱上,按前述的条件分离。产物TNT-1或F(ah′)_2的免疫活性,用标记抗体和过量抗原结合的方法测定;分高纯化过程中的洗脱峰,用间接免疫荧光技术监测其活性。结果戾明,TNR-1的F(ab′)_2片段的制备是成功的,色层分离纯化的方法简便而有效,并已成功地应用于批量生产。
TNT-1 is a murine IgG2a monoclonal antibody which has been used for clinical patients trials in patients. This paper describes methods for further purification of TNT-l from patients ascite and preparation of F(ab')2 fragments of TNT-1. The IgG was separated from their ascite by protein A affinity chromatography, and then further separated by cation exchange chromatography (Mono S column) on FPLC system. Digestion of TNT-l with pepsin was optimized and performed at pH 3.8. pepsin/antibody ratio of 1/250, and 4~ 5 hours of incubation at 37℃ The F(ab')2 fragment was purified by protein A and Mono S columns procedures using FPLC system. Kinetics of digestion and protein purity were studied by using superose 12 FPLC and SDS-PAGE. Immunoreactivities were determined by both methods of indirect immunofluorescence and direct radioimmunoas-say. The results revealed that the chromatographic procedures are practial, simple, and effective, and have been successfully used to produce gram quantities of clinical grade F(ab')2 fragment for the diagnosis of cancer in patients.
出处
《中山医科大学学报》
CSCD
1991年第4期311-315,共5页
Academic Journal of Sun Yat-sen University of Medical Sciences
关键词
单克隆抗体
TNT-1
免疫活性
Monoclonal antibody TNT-1 F(ab')_2 Purification Immunoreactivity
