Identification of a target antigen recognized by a mouse monoclonal antibody to the bile canalicular surface of rat hepatocytes with a random phage display library.

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.

Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

Anti-idiotype antibodies (Ab2) play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR) is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH). We previously developed a mouse monoclonal antibody (clone 8D7) which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1). One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

Anti-asialoglycoprotein receptor autoantibodies, detected by a capture-immunoassay, are associated with autoimmune liver diseases.

In autoimmune chronic active hepatitis (AIH) and primary biliary cirrhosis (PBC), various autoantibodies including anti-asialoglycoprotein receptor (ASGPR) antibodies have been found in patients' sera. We have previously developed a mouse monoclonal antibody against rat and human ASGPR. In this study, we developed a capture enzyme-linked immunosorbent assay (ELISA) for detection of anti-ASGPR antibodies using this monoclonal antibody and investigated the occurrence of anti-ASGPR antibodies in the sera of patients with various liver diseases. Serum samples were obtained from 123 patients with various liver diseases, including 21 patients with AIH and 40 patients with PBC. In this capture ELISA, the target antigen in the crude rat liver membrane extracts was captured on the ELISA wells by the ASGPR-specific mouse monoclonal antibody. Thus, the cumbersome process of antigen purification was rendered unnecessary. Using this capture ELISA, we detected the anti-ASGPR antibody in 67% of the patients with AIH, in 100% of the patients with PBC, and in 57% of the patients with acute hepatitis type A. However, the anti-ASGPR antibody was rarely detected in patients with other liver diseases such as primary sclerosing cholangitis and obstructive jaundice. Our findings suggest that this capture ELISA would be useful for the detection of anti-ASGPR antibodies in autoimmune liver diseases.