Purification of an anti-HBsAg scFv and measurement of its affinity constant
- VernacularTitle:重组人源抗HBsAg单链抗体的纯化及亲和常数测定
- Author:
Sheng XIONG
;
Xiangrong REN
;
Xing YAN
;
Yonghong TANG
;
Yehua ZHENG
;
Kuanyuan SU
;
Zhouyao YU
;
Ruhu YAO
- Publication Type:Journal Article
- Keywords:
Hepatitis B;
Antibodies;
Inclusion;
Antibody affinity
- From:
Chinese Journal of Pathophysiology
1999;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6?His tag, and to determine the affinity constant of the purified recombinant product.METHODS: Solubilizing in buffers containing urea or guanidine hydrochloride (GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni 2+ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA. RESULTS: The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached (61.08?1 45)%. The affinity constant of the polished scFv was confirmed to be(2.30?0.32) ?10 7 L/mol. CONCLUSION: The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro . The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.
