Merck + Co., Inc.

Edward H Wasmuth

William J McAleer

William J Miller

Roy A Machlowitz

An improved process for the preparation of hepatitis B antigen by subjecting plasma of hepatitis B donors to isopycanic banding wherein the step gradient is NaBr. Faster and more economical processing is obtained by multiple loading of the step gradient, either NaBr or conventional gradients.

Hepatitis B antigen

Compositions comprising HB.sub.s Ag and a physiologically acceptable medium are useful as diagnostic and immunologic agents.

Hepatitis B core antigen composition

Hepatitis B e antigen or hepatitis B e antibody are separated from human biological fluid by centrifugation in a density gradient and isolated in the 1.04-1.15 g/cc density region.

Isolation of hepatitis B e antigen

Hepatitis B surface antigen free of gradient-derived added cesium and potassium ions, for example the antigen in an aqueous solution of sodium bromide.

High purity HB.sub.s Ag is injected into guinea pigs to stimulate formulation of HB.sub.s Ab-positive serum. Gamma globulins containing HB.sub.s Ab are separated from the serum and the globulin fraction containing HB.sub.s Ab is isolated and coupled to a Sepharose gel. HB.sub.s Ag from HB.sub.s Ag-positive plasma is adsorbed on the gel by formation of an HB.sub.s Ab-HB.sub.s Ag complex. HB.sub.s Ag is eluted from the column and dialysed.

Isolation of HB.sub.s Ag

Hepatitis B immune globulin is prepared from individuals who exhibit an increase in hepatitis B surface antibody of at least 2,000 PHA units/ml following immunization with hepatitis B surface antigen.

Method for preparing hepatitis B immune globulin

Biological fluid containing hepatitis B surface antigen is subjected to isopycnic banding with collection of fractions rich in Dane particles. The Dane particles are useful as diagnostic and immunologic agents.

Isolating hepatitis B Dane particles

An improved process for the preparation of hepatitis B antigen by subjecting biological fluid containing heptatitis B surface antigen to isopycanic banding wherein the step gradient is NaBr. Faster and more economical processing is obtained by multiple loading of the step gradient, either NaBr or conventional gradients.

HB.sub.c Ag is prepared by isolating Dane particles by isopycnic banding of fluid from HB.sub.s Ag positive donors, optionally but preferably pelleting the Dane particles, and then removing the surface antigen by contacting the Dane particles with a nonionic surfactant having from about 15 to about 35 oxyethylene units in the presence of a reducing agent such as mercaptoethanol. The isolated core antigen is used as a diagnostic and immunologic agent.

Method of preparing hepatitis B core antigen

Trachoma agent is separated from growth medium impurities by density gradient centrifugation in a sodium bromide solution.

Separation of trachoma agent from growth medium impurities

Biological fluid containing hepatitis B surface antigen is concentrated using ammonium sulfate, and subjected to an isopycnic banding followed by rate zonal banding.

Hepatitis B surface antigen

Sensitizing plasma protein is dissociated from antigen-plasma protein complex by agitating at low temperature under controlled pH.

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Telford, PA, United States of America

Edward H Wasmuth