Purification of antigens from hydatid cyst fluid by molecular sieve chromatography
Objective To optimize the method for preparation of hydatid cyst fluid antigen by purifying crude antigen to remove nonspecific reactive proteins.Methods Curde antigen was obtained from sheep hydatid fluid by ultra filtration,and analyzed by Western blotting to identify nonspecific immunoreactive bands.For purification,molecular sieve chromatography with Superdex GL column was used to remove primary nonspecific reactive proteins.The purified antigen was evaluated for diagnostic efficacy using immunoblotting and ELISA with the sera of 44 cystic echinococcosis (CE) patients,17 healthy subjects and 10 cysticercosis patients.Resluts A primary nonspecific proteins band of Mr 55 000 was identified by Western blotting probed with infected and normal sera.After purification,immunoblotting demonstrated that the nonspecific component was removed from the crude antigen successfully.The sensitivity,specificity,Yourdon's index,and cross-reaction with cysticercosis of the purified antigen in ELISA were 93.2％ (41/44),94.1％ (16/17),0.87 and 30％ (3/10) respectively.The same parameters of the curd antigen were 90.9％ (40/44),88.2％ (15/17),0.79,and 60％ (6/10) respectively.The sensitivity of purified antigen was significantly higher than the curd antigen.Conclusion Crude antigen of sheep hydatid cyst fluid was purified by molecular sieve chromatography,and a Mr 55 000 nonspecific immunoreactive protein was identified and removed.The purification method used in this study provides clues for optimizing preparation of cyst antigen and quality control in development of effective diagnostics for echinococcosis.
NETL
NSTL
万方数据
维普
