AN6520 Ag: An antigen purified from liver with non‐A, non‐B hepatitis

Abstract

An extract prepared from the liver of patient with chronic non-A, non-B (NANB) hepatitis was found to produce a precipitin line in immunodiffusion with a serum from a multiply transfused patient and those from patients convalescent from NANB hepatitis. The antigen was purified by gel filtration and density gradient centrifugation. The antigen had a buoyant density of 1.16-1.20 g/cm3 in cesium chloride, a sedimentation coefficient (S20,w) of 51.5, and a molecular weight of larger than 1.5 X 10(6) daltons. Electron microscopic examination revealed particles 29-34 nm in diameter (average 31.5 nm), which could be agglutinated by the specific antiserum. We developed a reverse passive hemagglutination (R-PHA) and a passive hemagglutination (PHA) technique for detection of the new antigen and antibody, respectively, and applied these to human sera. Antibody to the antigen was detected in 19/28 (67.9%) convalescent sera of NANB hepatitis. This prevalence was significantly higher than those found in convalescent sera of type A hepatitis patients (2/17 = 11.8%), type B hepatitis patients (2/15 = 13.3%), and normal blood donors (9/129 = 7.0%) (p less than 0.01); and the prevalence in hepatitis A and B patients did not differ significantly from that of normal donors. Furthermore, most (66.7%) of the cases of NANB hepatitis endemic in Shimizu City, Japan, showed clear seroconversion with respect to this antibody. These results suggest that the new antigen/antibody system is associated with NANB hepatitis.

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A

“…We purified the antigen and developed passive hemagglutination assay (PHA) using antigen-coated erythrocytes to detect antibody in patients' sera. We found that the antigen is composed of particles with molecular weight of more than 1.5 × 10 6 Da and diameter of 29-34 nm. The antibody was detected in 37.5% in NANBH cases, but not in control groups [6].…”

“…We found that the antigen is composed of particles with molecular weight of more than 1.5 × 10 6 Da and diameter of 29-34 nm. 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].…”

“…Active fractions were pooled, concentrated using Centriplus YM-10 (Millipore, Bedford, MA), and then dialyzed against buffer A containing 0.05% Triton X-100. Membrane-bound form of mEH was purified according to the method for AN-Ag [6]. The homogenate from 1.2 g of infected SF9 cell pellet was applied onto a Sephacryl S-300 (Amersham, Uppsala, Sweden) column (2.5 × 90 cm) and eluted with TBS containing 1 mM EDTA (buffer C).…”

“…A λgt11 cDNA library from human liver (Clontech, Pal-oAlto, CA) was screened with rabbit anti-AN antibody [6] (50 μg/ml), by the standard protocol [15]. After four cycles of selection, they were further selected by using their phage lysates for affinity purification [16] of rabbit anti-AN antibody followed by incubation of the eluted antibody on the immunoblot of mEHcontaining cell lysate.…”

“…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).…”