Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant

To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1. The specific anti-ASGPR phage clone C1 was transfected into E. coli HB2151. The single colony was chosen to be inoculated i...

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Published inXi bao yu fen zi mian yi xue za zhi Vol. 22; no. 4; p. 504
Main Authors Wang, Wei-yu, Yi, Ji-lin, Deng, Yun-hua, Si, Jin, Wang, Cong-jun, Li, Xiang, Cao, Li-min
Format Journal Article
LanguageChinese
Published China 01.07.2006
Subjects
Antibodies, Monoclonal - analysis
Antibodies, Monoclonal - biosynthesis
Antibodies, Monoclonal - immunology
Antibodies, Monoclonal - isolation & purification
Antibody Affinity
Asialoglycoprotein Receptor - immunology
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Escherichia coli - genetics
Gene Expression
Genetic Therapy
Humans
Immunoglobulin Variable Region - analysis
Immunoglobulin Variable Region - biosynthesis
Immunoglobulin Variable Region - immunology
Immunoglobulin Variable Region - isolation & purification
Liver Neoplasms - genetics
Liver Neoplasms - therapy
Temperature
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Summary:To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1. The specific anti-ASGPR phage clone C1 was transfected into E. coli HB2151. The single colony was chosen to be inoculated into 2 x TY medium and shaken (250 r/min) overnight at 37 degrees C. After 1 in 100 dilution in 2 x TY medium and induced for secreted expression, scFv C1 was induced at different concentrations (0.25, 0.5 and 1.0 mmol/L IPTG) overnight at 37 degrees C, 25 degrees C and 20 degrees C, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30 mL PBS and dialyzed against PBS overnight at 4 degrees C. The expressed scFv C1 was purified by Ni(2+) chelating HiTrap HP column and the purity of the purified scFv C1 was identified by SDS-PAGE. Then affinity constant of scFv C1 was determined by noncompetitive enzyme
ISSN:1007-8738