The United States of America as Represented by the Department of Health

Berge Hampar

Stephen D Showalter

Martin Zweig

Conrad J Heilman, Jr

Russell H Neubauer

Harvey Rabin

Ralph F Hopkins, Iii

A method and test kit for the serological diagnosis of human infection by herpes sipmlex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) using immunoaffinity purified virus-coded glycoproteins as target antigens. A preferred embodiment of the method employs a variation of the enzyme-linked immunosorbent assay (ELISA) whereby monoclonal antibodies are used to purify target antigens, and test sera are absorbed with virus-infected cell extracts to remove intertypic cross-reacting antibodies.

Enzyme-linked immunosorbent assay (ELISA) for determining anti-bodies

A method for producing monoclonal antibody reagents against novel proteins induced by herpes simplex virus type 1 (HSV-1). The method consists of preparing HSV-1 antigen populations by infecting mammalian cells either with HSV-1 alone or with HSV-1 in the presence of an inhibitor of protein synthesis, allowing virus replication to proceed by reversing the action of said inhibitor, inoculating said antigen mixture in mice to induce the production of antibodies, fusing the spleen cells of said mice with myeloma cells to obtain hybrid cells, and screening said cells by radioimmunoprecipitation-polyacrylamide gel electrophoresis (RIP-PAGE) to identify hybrid cells producing monoclonal antibodies against HSV-1 proteins. The method teaches the production of unique monoclonal antibody reagents directed against novel HSV-1 proteins; including a 132,000 molecular weight (mw) DNA-binding protein, a 175,000 mw immediate-early protein, and a previously unknown 110,000 mw glycoprotein.

Monoclonal antibodies to herpes simplex virus type I polypeptides

A non-transformed cell line is produced by treating BALB/c derived 10E2 cells with 5-bromodeoxyuridine and 5-iodo-2'-deoxyuridine to produce thymidine kinaseless cells which upon multiple cloning show a flat epitheloid appearance indicative of their non-transforming potential. These cells are used to determine the tumorigenic transforming potential of any gene by introducing the gene into the cells of the non-transformed cell line along with the Herpes simplex virus thymidine kinase gene which serves as a vehicle for cotransfection. The transformation of the cells is indicative of tumorigenic potential.

Non-transformed thymidine kinaseless cell line and its use for testing

The method of producing clinical assays for use of monoclonal antibodies in the diagnosis of Herpes simplex virus (HSV) infections and the differentiation of Herpes Simplex virus types 1 and 2 as a diagnostic kit for differentiating HSV-1 and HSV-2 utilizing clone 1D4 against HSV-1 and clone 3E1 against HSV-2.

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Middletown, MD, United States of America

Berge Hampar