Electrofocusing of Hepatitis B Antigen

Howard, Colin R.; Zuckerman, Arie J.

doi:10.1099/0022-1317-20-2-253

Cited by 13 publications

(6 citation statements)

References 2 publications

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“…the filaments (4.1), and the Dane particles (3.82), although it is probably not related to particle size. Howard and Zuckerman [19] recently reported the presence of two unlabeled populations, with pH values of 3.65 and 4.33, in HBsAg prep arations partially purified by gel chromatography in Sephadex G-200. These preparations ranged in size from 20 to 25 nm.…”

Section: Discussionmentioning

confidence: 99%

Biophysical Properties of Purified Morphologic Forms of Hepatitis B Antigen

Chairez¹,

Fb²,

Jl³

et al. 1974

Intervirology

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The morphological forms of hepatitis B antigen (HBsAg) present in an HBsAg-positive plasma (15–25 nm spheres, filaments 20–22 nm in diameter and 80–300 nm in length, and 42 nm Dane particles) were isolated and characterized biophysically. The HBsAg in this plasma was fractionated into five distinct populations, containing predominately 15–19 nm spheres, 20–22 nm spheres, 20 × 300 nm filaments, a mixture containing 23–25 nm spheres and rare filaments, and 42 nm Dane particles, respectively. These populations were labeled with 125I and electrofocused in a pH ampholyte gradient. Each population had a distinct isoelectric pH with values ranging from 3.65 to 4.70. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of each of the above labeled populations revealed the presence of 7, 10, 9, 5, and 9 polypeptides, respectively. The population containing predominately 23–25 nm spheres failed to react with antibody specific for HBsAg and probably was composed of inner cores of the Dane particles.

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Section: Discussionmentioning

confidence: 99%

Biophysical Properties of Purified Morphologic Forms of Hepatitis B Antigen

Chairez¹,

Fb²,

Jl³

et al. 1974

Intervirology

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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: 85%

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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“…Partially purified hepa titis B antigen, separated from serum proteins by gel filtration, may be resolved into at least two populations of intact morphological forms, according to surface charge [21], Hepatitis B antigen separated as described above and trace-labelled by the chloramine-T method resolves the two peaks over a higher pH range ( fig. 6).…”

Section: Resultsmentioning

confidence: 99%

“…Of interest is the observation that antigenic activity was not detected in association with separated y-globulins, which is in accord with the long epidemiological and clinical experience that y-globulin is free of the risk of transmitting hepatitis and with the failure to detect hepatitis B antigen, a marker associated with infectivity, by electron microscopy after Cohn fractionation of human plasma known to contain the antigen [28]. We have previously shown that if the major portion of serum proteins was removed by gel filtration, the technique of electrofocusing separates the antigen from the remaining un wanted serum proteins and reveals a heterogeneity in the surface properties of the small antigen particles [21]. The isoelectric points of the two isolated bands were found to differ according to the nature of the sub-determinants and to share at least one common antigenic determinant as demonstrated by immune electron microscopy [22], In a further series of experiments, purified hepatitis B antigen was iodinated with the radioisotope 125I after separation from serum proteins by polyethylene glycol precipitation and equilibrium centrifugation in cesium chloride.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Hepatitis B Antigen Polypeptides

Howard¹,

Zuckerman²

1974

Intervirology

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The close association of hepatitis B antigen with serum proteins was demonstrated. The small morphological form of the antigen, measuring approximately 20 nm in diameter, was separated by isoelectric focusing and the heterogeneity of the antigen was confirmed by the finding of unique polypeptides in each preparation. Comparative trace-labelling techniques showed the integral nature of the constituent polypeptides, two of which were found to be mutually exclusive in the separated particles. Release of these polypeptides was achieved by treatment with Nonidet P40 and 2-mercaptoethanol. The significance of these findings in relation to hepatitis B virus is discussed together with the importance of surface antigenic variation as a function of protein heterogeneity of this virus.

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Electrofocusing of Hepatitis B Antigen

Cited by 13 publications

References 2 publications

Biophysical Properties of Purified Morphologic Forms of Hepatitis B Antigen

Biophysical Properties of Purified Morphologic Forms of Hepatitis B Antigen

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

Characterization of Hepatitis B Antigen Polypeptides

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