Abstract

This study screened specific single-chain variable fragment (scFv) antibody against asialoglycoprotein receptor (ASGPR) by capture phage enzyme-linked immunosorbent assay (ELISA). The CRDH1/pET3b plasmid containing ASGPR gene was employed to amplify the CRDH1 gene of subunit H1 of ASGPR through PCR, which was then directionally cloned into prokaryotic expression vector pET-32c. The expression of CRDH1 was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the expression products underwent affinity purification through a Ni^2+-chelating column. The humanized phage antibody library was screened and identified by capture phage ELISA. Then, the selected CRDH1 single chain antibody was sequenced, expressed, purified and identified by Western blot and compared with the antibody from indirect phage ELISA.  The recombinant CRDH1 protein was a 35 kDa fusion protein which existed as an inclusion body. Affinity purification through a Ni^2+-chelating column acquired the CRDH1 fusion protein. 44 colonies out of 60 colonies could specifically bind to CRDH1. Sequencing showed that 9 colonies had difference sequences and 9 colonies could express CRDH1 specific scFv. Furthermore, the purified scFv also specifically recognized rCRDH1. Capture phage ELISA effectively improved the GST fusion proteins, causing false positives and enhanced the positive rate. In addition, 9 rCRDH1 specific scFv antibodies were successfully obtained by capture phage ELISA.

Key words: Asialoglycoprotein receptor, enzyme linked immunosorbent assay, fusion protein, single-chain variable-fragment antibody.