Benefit and application of antibodies against the H1 carbohydrate recognition domain of the human hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR), composed of the subunits H1 and H2, is predominantly expressed on the sinusoidal surface of mammalian hepatocytes and involved in the endocytosis of desialylated multiantennary glycans with terminal Gal or GalNAc residues via the clathrin-coated pit pathway. Although investigated for many years, the physiological function and ligands are still unknown. Until nowadays, only polyclonal antibodies and two monoclonal against the ASGPR are reported. However, no monoclonal antibodies, which are specifically directed against the carbohydrate recognition domain (CRD), responsible for Gal binding and internalization, are available. Such antibodies would be precious tools for ASGPR examination. In addition, since the human ASGPR is a promising liver-specific therapeutic target, an antibody-based high affine and specific drug or gene delivery system would be valuable. The H1-CRD was recombinantly expressed in E.coli and purified active H1-CRD was used for immunization to produce antibodies. Because the published expression and purification method of Meier et al.26 yielded only an H1-CRD amount of 130 g/L culture, various E.coli strains, vectors, expression conditions and renaturation procedures were tested. With the optimized expression in E.coli AD494(DE3), which was transformed with the H1-CRD cDNA-encoding pET3b vector, and with the following modified purification procedure, 55mg (20mg/L) active H1-CRD were successfully produced. For analytical purpose, polyclonal antibodies directed against the H1-CRD (anti-H1) were obtained by chicken immunization and IgY purified from egg yolk by PEG precipitation. The 12g (45mg/egg) purified total IgY contained approximately 7% anti-H1 specific IgY. Total IgY and isolated anti-H1 specific IgY antibodies showed to be suitable in various immunochemical in vitro methods but not in immunocytochemistry. Although the amino acid sequences of the mouse and human ASGPR H1-CRD are 79% identical, monoclonal antibodies were successfully produced by the hybridoma technology. Eight of twenty hybridomas were selected and cloned for further characterization of their monoclonal antibodies: in vitro in immunoblotting, immunoassays, Biacore assays and in epitope mapping, on cell in flow cytometry and fluorescence microscopy and in tissue in immunohepatohistochemical tests. Two of them, C14.6 and particularly C11.1 showed a very interesting profile, not only for application in immunochemical techniques but probably also for diagnostic and therapeutic use.