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Immunochemistry of the Hepatitis B Virus: ¹²⁵I HB Ag Ligand¹

Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037, and the Rockville Laboratory of the Molecular Anatomy (MAN) Program, Oak Ridge National Laboratory, Rockville, Maryland 20852

Abstract

Immunochemical studies of the Hepatitis B virus and its gene products require labeled virus or derivative gene products of high antigenic and structural integrity. Such ligands can be provided by ¹²⁵I Hepatitis B antigen (HB Ag). Techniques for the iodination of microgram quantities of 22 nm HB Ag particles with either immobilized lactoperoxidase or chloramine-T are described. Specific activities of 2 to 10.0 µCi/µg can be achieved. The labeled products of both methods compare favorably with unlabeled antigen in terms of sedimentation velocity and density; however, the solid-phase lactoperoxidase method produces an ¹²⁵I HB Ag ligand of superior antigenic integrity. ¹²⁵I HB Ag prepared by lactoperoxidase has an in vivo clearance rate (T) of 23 hr as compared to 19 hr for chloramine-T-labeled ¹²⁵I HB Ag. Competitive inhibition radioimmunoassays, with ¹²⁵I HB Ag, are influenced by the antigenic integrity of the ligand and only with the lactoperoxidase product can concentrations as low as 10 ng/ml (3 x 10^-12 M) of purified unlabeled 22 nm HB Ag particles be detected. Deterioration of trichloroacetic acid precipitability and immunoprecipitability of ¹²⁵I HB Ag is observed over a period of weeks, which imposes a requirement for fresh ¹²⁵I HB Ag if maximum antigenic integrity of the ligand, sensitivity, and immunochemical precision are to be achieved.

Footnotes

¹ This is Publication 817 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California. These studies were supported by National Institutes of Health Contract NIAID-73-2509. The Rockville Laboratory of the MAN Program is supported by the NIAID under Union Carbide's Contract with the United States Atomic Energy Commission.

² Address reprint requests to Francis V. Chisari, M.D., Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037.

³ Present address: Molecular Anatomy Program, Oak Ridge National Laboratory, Rockville, Maryland 20852.

⁴ Present address: Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037.