COMPARATIVE STUDIES ON PURIFICATION OF ENGINEERED ANTI-HBSFAB WITH 3 DIFFERENT METHODS / 解放军医学杂志

ABSTRACT

The purpose of this study was to compare the efficiency of 3 different methods in purifying engineered anti-HBsFab. Anti-HBsFab Supernatants containing anti-HBsFab were prepared by ultrasound lysis of host cells of anti-HBsFab positive clone. Anti-Fab-sepharose gel, streptococcal protein G-sepharose gel (Prot G gel) and Ni-NTA-agarose gel were used to purify Fabs according to the reagents protocols. Then the concentrations of purified proteins were determined. SDS-PAGE was used for the measurement of purified Fabs'purity. HBsAg based ELISA was chosen to determine the Fabs' bio-activity. The results indicated that Fab recovery of different gels in equal volume were different. The recovery of Ni-NTA GEL was greater than that of Prot G gel, and the recovery of Prot G gel was greater than that of anti-Fab gel. Purity of the Fab isolated by 3 different gels was confirmed by SDS-PAGE. The binding capacity of anti-HBs Fab to HBsAg of these three gels had no signifticant difference. Our data suggested that each method had its advantage and usage limitation, Ni-NTA method demonstrated much better performance in economy, management, and efficiency over the other two methods.