Characterization of a single-chain variable fragment (scFv) antibody directed against the human asialoglycoprotein receptor

To obtain scFv (single-chain variable fragment) against human ASGPR (asialoglycoprotein receptor), a human non-immune phage antibody library was screened with the recombinant CRD (carbohydrate recognition domain) of rCRDHI (the HI subunit of human ASGPR). Anti-rCRDHI phage clones were obtained after four rounds of screening with rCRDHI-coated immunotubes and single positive colonies were further selected with the expressed thioredoxin-His-S tag of the wild-type pET32c. Two specific anti-rCRDHI phage clones (named C1 and C2) were transfected into Escherichia coli HB2151 and induced for secreted expression of scFv antibody. The purified anti-rCRDHI C1and C2 single-chain antibodies were characterized by immunoblotting and immunohistochemistry. Both antibodies were found to specifically recognize denatured and native forms of the ASGPR and thus could potentially be used as targeting molecules for gene therapy of hepatocellular carcinoma or other liver diseases.