Study of structural and antigenic components of Hepatitis B surface antigen
Although the spread of hepatitis B infection has largely been controlled through the use of sophisticated serological tests for the detection of hepatitis B surface antigen there is still comparatively little basic inf creation available on the protein, lipid and carbohydrate composition of these particles, and on the arrangement of these components into a stable quaternary structure. In the present work three separate approaches have been made to extending this information. 1. Polypeptide analysis of purified HB_Ag particles by column 0 chromatography and discontinuous or continuous polyacrylamide gel electrophoresis (PAGE) produced discrepant polypeptide profiles; in the latter system reaggregation of protein constituted a major problem, and with all techniques incomplete dissociation and/or reaggregation during handling and storage of antigen were noted. In the discontinuous PAGE system one major polypeptide of 14,000 daltons or less and one minor polypeptide of 60-70,000 daltcms 125 were consistently detected in both unlabelled and I labelled HBgAg. However, major limitations in the use of standard PAGE techniques for the analysis of HB_Ag polypeptides were clearly 0 documented, which may contribute to the present confused situation in the literature relating to the number, molecular weight and immunogenicity of these components.2. 22nm HB Ag particles were treated with a range of denaturing agents including nonionic or anionic detergents, reducing agent, or high concentrations of urea or guanidine hydrochloride. The particle displayed considerable resistance to disruption under strong denaturing conditions, in one instance only incomplete dissociation to a stable subunit of 25S being achieved after boiling with sodium dodecyl sulphate and reducing agent. However, prolonged incubation with hi$i concentrations of Triton X-100 released a polypeptide of 70-80»000 daltons which retained both group specific a and subtype specific d activity when analysed by double antibody radioimmunopreoipitation using monospecific antisera. 3. Examination of HBaAg particles by electron microscopy following treatment with denaturing agents revealed a number of stages in the breakdown of these structures. The smallest detectable morphological structures were ring shaped units of 4-6nm diameter; a model for the assembly of these units into the complete 22nm HB_Ag particle is proposed.