Heterogeneity of hepatitis B surface antigen-associated particles isolated from chimpanzee plasma

Abstract

Hepatitis B surface antigen (HBsAg) was purified from approximately 8 liters of pooled plasma from a carrier chimpanzee. Precipitation of HBsAg with polyethylene glycol resulted in more than 20-fold purification, with about 80% recovery of antigenic activity. The sample was separated by further purification and fractionation into three populations of HBsAg-associated particles by column chromatography on hydroxylapatite: the first contained short filaments and 22to 28-nm spheres, the second was composed of larger filaments and variable-sized spheres, and the third contained mostly 16to 22-nm spherical particles. A large volume of the polyethylene glycol precipitate passed through hydroxyl-apatite twice yielded over 650 mg of partially purified HBsAg. A pooled preparation of purified HBsAg was separated by zone-convection electrofocusing into five peaks of antigenic activity within the pH range of 4.7 to 5.7.The chimpanzee has been shown to be susceptible to infection with hepatitis B virus and may develop either a transient or chronic carrier state (1,7,13,14,16,17). Hepatitis B surface antigen (HBsAg) isolated from chimpanzee plasma closely resembles that isolated from human sources morphologically and serologically (6, 12).We report herein a modification of the polyethylene glycol (PEG) procedure of De Rizzo et al. (5) for the isolation of HBsAg from chimpanzee plasma. It is shown that column chromatography on hydroxylapatite separates the isolated HBsAg-associated particles into populations differing in size distribution and isoelectric points. MATERIALS AND METHODSSource of HBsAg. HBsAg of the adw subtype was purified from pooled plasma of a carrier chimpanzee previously inoculated with plasma from a chronic carrier chimpanzee.HBsAg was isolated by PEG precipitation. The resuspended PEG precipitate of HBsAg was submitted to fractionation and purification by column chromatography on hydroxylapatite. The methods of purification were modifications of those described previously (23).Precipitation of HBsAg by PEG 6000. In a prepurification step, 7,800 ml of pooled HBsAg-containing plasma was adjusted to pH 4.6, and a 30% solution of PEG (Union Carbide Co.) in distilled water was added to a final concentration of approximately 2%. The suspension was stored overnight at 4°C and clarified by centrifugation. The HBsAg in the supernatant was precipitated by raising the PEG concentration to 4%, and the antigen was separated by centrifugation after overnight storage of the suspension at 4°C. The sediment of HBsAg was resuspended to 500 ml in distilled water. The bulk of PEG and some accompanying contaminants were precipitated by adjusting the solution to pH 5.0. The sample was stored at 4°C overnight and clarified by centrifugation. The clear supernatant was readjusted to pH 4.6, and the HBsAg was precipitated by adding a 30% solution of PEG to a final concentration of 4%. The suspension was again stored overnight at 4°C, and the HBsAg was separated by centrifugation. Finally, the sediment of HBsAg was resuspended to 320...