Detection of an Antigen (AN6520), Possibly Related to Non‐A, Non‐B Hepatitis, by Monoclonal Antibodies. I

Abstract

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.

Microsomal epoxide hydrolase 1 (EPHX1): Gene, structure, function, and role in human disease

“…Importantly for cancer biomarkers, Coller et al (2001) suggested that immunohistochemical detection of EPHX1 may be a useful diagnostic tool for hepatocellular carcinoma. Localization of EPHX1 in the membrane changes during liver pathogenesis, e.g., neoplasia (Gill et al 1983) or hepatitis infection (Akatsuka et al 1986) and is often accompanied by aberrations of EPHX1 structure (Akatsuka et al 2007). EPHX1 specifically binds hepatitis B spliced protein (HBSP) and enhances the carcinogenic effect of benzo[a]pyrene in vitro (Chen et al 2010 and 2014).…”

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

“…The appearance of mEH in the blood is also associated with other types of liver disease (Hammock et al , 1984). In hepatitis C infection, we have shown that the hepatitis C-associated antigen (AN antigen) appears in the early phase of the viral infection (Akatsuka et al , 1986b), which is followed by the development of the antibody in the acute phase of hepatitis (Akatsuka et al , 1986a). In a recent study, we have shown that the AN antigen is mainly composed of mEH (Akatsuka et al , 2007).…”

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A

“…Rabbit anti-AN antibody and mouse anti-AN mAb 1F12 have been described [6,7]. Rabbit anti-mEH antibody was raised by injecting a rabbit (s.c.) with 400 μg of solubilized and purified mEH three times (first with Freund's complete adjuvant, second and third immunizations with incomplete adjuvant).…”

“…The antibody was detected in 37.5% in NANBH cases, but not in control groups [6]. Then we developed monoclonal antibodies (mAbs) and used one of them, 1F12, to develop radioimmunoassays (RIAs) for the antigen and for the patient's antibody [7]. The antigen and antibody were detected in the acute phase and convalescent phase sera, respectively, of some patients with NANBH.…”

“…Toward isolating and identifying AN-Ag, we initially tried to use the RIA assays developed previously [7]. However, the inhibition RIA is not specific enough in that it cross reacted with an unknown protein present in the serum of many people.…”