title: Aggregation of recombinant hepatitis B surface antigen induced in vitro by oxidative stress.
abstract: In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic. During more gentle oxidation with 9 mM AP and 20-80 microM Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event. During this pre-aggregation period, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized particles. In contrast, yeast-derived HBsAg aggregate is decomposed to S monomers under reducing conditions and recognized by anti-HBsAg polyclonal and monoclonal antibodies, suggesting that is has been assembled in vivo from antigenic and reversibly cross-linked particles. Based on these observations, we conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation in vivo. Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidation in biological samples. Hence, at present, the magnitude of the in-vivo oxidative damage to HBsAg cannot be evaluated and thus, whether the plasma-derived HBsAg undergoes radical-induced oxidation in the course of viral hepatitis remains to be established. If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood donors and inconsistency in the results provided by HBsAg- and anti-HBsAg-based tests in several recent reports.
authors.affiliation: Quality Control Department, National Center for Bioproducts, Havana, Cuba.
authors.forename: D
authors.initials: D
authors.lastname: Tleugabulova
authors.forename: V
authors.initials: V
authors.lastname: Falcón
authors.forename: E
authors.initials: E
authors.lastname: Pentón
authors.forename: M
authors.initials: M
authors.lastname: Sewer
authors.forename: Y
authors.initials: Y
authors.lastname: Fleitas
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007118:Immunoassay; D016253:Microscopy, Immunoelectron; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010843:Pichia / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; C031276:ammonium peroxydisulfate; D000645:Ammonium Sulfate