Generation of monoclonal antibodies directed against the immunogenic glycoprotein K8.1 of human herpesvirus 8.
Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.
Lang
D
D
Biotest AG, Dreieich, Germany. dieter_lang@biotest.de
Birkmann
A
A
Neipel
F
F
Hinderer
W
W
Rothe
M
M
Ernst
M
M
Sonneborn
H H
HH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody / Q000276:immunology; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017931:DNA Primers; D000939:Epitopes; D006023:Glycoproteins; C113454:K8.1 protein, Human herpesvirus 8; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase