Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw

Chairez, Ruben; Hollinger, F. Blaine; Brunschwig, J. P.; Dreesman, Gordon R.

doi:10.1128/jvi.15.1.182-190.1975

Cited by 29 publications

(8 citation statements)

References 18 publications

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“…The molecular-weight estimates of the polypeptides described in this report are in reasonably good agreement with the major ones described earlier (13) and by others (6,34). We have been unable to detect antigenic activity in HB,Ag components other than the P-1 through P-7 polypeptides, in contrast to the reports of others (11, 26).…”

Section: Duressupporting

confidence: 92%

Proteins of hepatitis B surface antigen

Shih¹,

Gerin²

1977

J Virol

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Purified 22-nm forms of hepatitis B surface antigen (HBsAg) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might be glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBsAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBsAg yielded patterns identical to those obtained with protein stain. Comparison of

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Section: Duressupporting

confidence: 92%

Proteins of hepatitis B surface antigen

Shih¹,

Gerin²

1977

J Virol

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“…The heterogeneity of human HBsAg has been reported by many workers (2,3,8,9,26). Chairez et al (3) found three to four peaks of antigenic activity varying between pH 4.5 to 5.4 for unpurified, and pH 3.9 to 4.9 for highly purified, samples of both adw and ayw subtypes of human HBsAg.…”

Section: Discussionmentioning

confidence: 87%

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

Vnek¹,

Ikram²,

Prince³

1977

Infect Immun

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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...

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“…CHAIREZ et al [4], using P-mercaptoethanol and guanidine for reducing CsCl purified HB,Ag, have described polypeptides ranging from 19,000 to 120,000 molec. wt.…”

Section: Discussionmentioning

confidence: 99%

Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80¹

et al. 1976

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A simple, inexpensive procedure for the isolation and purification of HB8Ag from plasma is described. The technique included precipitation of HBsAg with PEG and elimination of normal plasma proteins by digestion with pepsin. Tween 80 was used to remove contaminating lipoprotein(s). This technique resulted in about a 200-fold gain in the specific activity of HBsAg and yielded about 20-40% recovery. Rabbits immunized with the purified antigen produced type-specific antibodies to HBsAg without detectable reactivity to normal human plasma antigens.

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Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw

Cited by 29 publications

References 18 publications

Proteins of hepatitis B surface antigen

Proteins of hepatitis B surface antigen

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

Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80¹

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Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw

Cited by 29 publications

References 18 publications

Proteins of hepatitis B surface antigen

Proteins of hepatitis B surface antigen

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

Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 801

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Product

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Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80¹