Immunochemical structure of the hepatitis B surface antigen vaccine--I. Treatment of immobilized HBsAg by dissociation agents with or without enzymatic digestion and identification of polypeptides by protein blotting.
Since the immunosorbent techniques and the cycles of isopycnic and rate zonal velocity ultracentrifugations were shown to be unsuitable for the purification of hepatitis B surface antigen (HBsAg) particles from human sera because HBsAg was still largely contaminated by serum proteins, we applied a drastic dissociating treatment of HBsAg stabilized by adsorption on silica gel which appeared essential to remove extraneous components initially present in the HBsAg particles. Only albumin and sometimes IgG were recovered with the purified antigen. The polypeptide composition of our purified HBsAg preparations was analyzed by SDS-PAGE with subsequent transfer to a nitrocellulose sheet by blotting, incubation with 125I-anti-HBs and exposure to X-ray film. Samples from HBsAg-positive sera containing the hepatitis B virus e antigen (HBeAg) displayed three proteins: P 24.5 and GP 28 as major components and GP 36 as a minor component. Dimers of these polypeptides were also immunologically detected. When a supplementary step of trypsin or pepsin digestion was included in our purification procedure after adsorption to silica and acid dissociation of HBsAg, proteolytic cleavage fragments of HBsAg with mol. wts lower than 10,000 were obtained on SDS-PAGE after reduction. This finding shows that arginine and lysine residues inaccessible to tryptic digestion in the intact HBsAg lipoprotein particle were exposed to enzymatic hydrolysis by our treatment. However, HBsAg kept the antigenic and immunogenic properties of the native antigen. Therefore such a HBsAg preparation appeared as a new candidate for the vaccination against HBV and a useful material for the analysis of the HBs antigenic structure.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006510:Hepatitis B Antibodies / Q000096:biosynthesis; D006514:Hepatitis B Surface Antigens / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008970:Molecular Weight; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D014357:Trypsin; D014765:Viral Vaccines / Q000032:analysis*
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D010446:Peptide Fragments; D010455:Peptides; D014765:Viral Vaccines; D014357:Trypsin; D010434:Pepsin A