Isolation and characterization of liver‐derived hepatitis B e antigen

Abstract

Two subpopulations of hepatitis B e antigen (HBeAg) were isolated from a human liver infected with hepatitis B virus. HBeAg extracted from liver homogenate subsequent to treatment with buffered 3 M NaSCN or 0.5 M MgCl2 banded at the density of 1.13 g/cm3 in CsCl and was polydispersed on gel filtration. In contrast, HBeAg released with phosphate-buffered saline (PBS) was detected mainly at a density of 1.20 g/cm3 in a CsCl gradient and consisted of low molecular weight species on gel chromatography. Polypeptides of 40,000 and 45,000 daltons were found in NaSCN and PBS-released HBeAg preparations, respectively. The results are interpreted as suggestive that liver HBeAg is a dimer of the major core particle polypeptide in different physicochemical forms.

Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus

“…Alanine scanning across this seven-residue segment showed no signi¢cant e¡ect on the ability of 5a19 to recognize the epitope (data not shown). The substitutions Arg35CGly, Thr38CSer and Ala39CAsn, corresponding to changes to the ad serotype, were also tried separately, but only the substitution at position 39 showed a reduction in recognition, consistent with the previously reported preference of 5a19 for the ay serotype [6].…”

“…The mAb 5a19 (isotype IgG1,U) was obtained by immunizing BALB/c mice with HBV particles of the ay serotype [6]. The immunoglobulin was puri¢ed from ascitic £uid by precipitation with 40% ammonium sulfate at pH 7.4, followed by ion-exchange chromatography on a DEAE-Sephacel (Pharmacia) column using an incrementing NaCl concentration gradient (0^500 mM) in Tris bu¡er at pH 8.0.…”

“…In particular, the preS1 peptide fragment comprising residues 21^47 (preS1(21^47)), as well as antibodies induced by this peptide inhibit HBV binding to HepG2 cells [4,5], implicating this region of the L envelope protein in the virus^host cell interaction. The murine monoclonal antibody (mAb) 5a19 recognizes a linear epitope preS1(36^43) [6] and inhibits invasion of cultured human hepatocytes [7]. Here, we present the sequence of the heavy and light chain variable domains (V H and V L , respectively), kinetic studies of antigen-binding, and ¢ne epitope mapping of the preS1 region recognized by 5a19.…”

Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization.

“…MHBs and MHBst were expressed in HeLa cells by a vaccinia virus/T7 polymerase system (see Materials and methods). Total cell extracts, a high speed supernatant and particulate fraction were analysed by Western blotting with the monoclonal antibody F-124 specific for the N-terminal half of the preS2 domain (Budkowska et al, 1986). Cells infected with T7 recombinant vaccinia virus alone did not give a specific signal (Figure 9a, lane 1).…”

Sequence analysis of a monoclonal antibody specific for the preS2 region of hepatitis B surface antigen, and the cloning, expression and characterisation of its single‐chain Fv construction

“…The antibody recognises an epitope that includes residue 126 of the preS2 region since it binds less well to the peptide segment 120^153 derived from the adw serotype, where Thr-126 from the immunising ay serotype is substituted by alanine [3]. Furthermore, the epitope includes the preS2 glycan N-linked to Asn-123 since removal of carbohydrate from HBsAg particles carrying proteins S and M sig-ni¢cantly reduces F124 binding [4]. Finally, the epitope is restricted essentially to the N-terminal region of preS2 since its binding to the peptide fragment 120^145 is not compromised by the presence of F376, a monoclonal antibody recognising the segment 132^145 [5].…”