FPLC纯化单克隆抗体（Fab＇）_2片段

Purification of Monoclonal Antibody F(ab')_2 Fragments by Using Fast Protein Liquid Chromatography on an Anion Exchange Column

作者采用ＤＥＡＥ－４０ＨＲ阴离子交换色谱柱，在Ｗａｔｅｒｓ６５０Ｅ快速蛋白液相色谱系统（ＦＰＬＣ）建立了抗肝癌单克隆抗体（ＨＡｂ１８）Ｆ（ａｂ＇）２片段的快速纯化方法。该法是将ＭｃＡｂ用胃蛋白酶消化１８ｈ后，上阴离子交换色谱柱纯化，用ｐＨ７．５１０ｍｍｏｌ／ＬＰＢ洗脱，即得到纯化的Ｆ（ａｂ＇）２．一次纯化周期仅需２５ｍｉｎ，一次上样量１２０～１６０ｎｇ蛋白，Ｆ（ａｂ＇）２得率为３４％，回收率为５１％。经ＳＤＳ－ＰＡＧＥ测定纯度大于９５％，免疫荧光法测定活性为１：４０００．该法操作简单、快速、实用性强，不需要高档次的色谱仪，只要有一台核酸／蛋白检测仪便可进行纯化，是实验室纯化Ｆ（ａｂ＇）２的理想方法．

The purification method of anti-hepatoma McAb HAbis F(ab')2 fragments has been established by using fast protein liqiud chromatography (FPLC) on a DEAE 40-HR anion exchange column. McAb HAb18 was digested at 37℃ for 18 hours hy Pepsin for which enzyme:IgG weight ratio was 1:25-33, and then it was purified on anion exchange column with PH7.5 10 mmol/L PB. The total protein sample loading; was 120-160 mg in an operating cycle of 25 min each time.The yield of F(ab')2 fragments was about 34%, and recoyery rate was 51%. The purity of F(ab')2 was over 95% by SDS-PAGE, and its immunoactivity was 1:4000(2. 72× 10-9mol/L) by fluorescent stainiug. So it is a practical method for purificotion of IgG F(ab')2 fragments in general labor-atory.