title: Expression of a human, neutralizing monoclonal antibody specific to puumala virus G2-protein in stably-transformed insect cells.
abstract: We cloned the heavy- and light-chain antibody genes of a human X (humanxmouse) trioma secreting a neutralizing, IgG monoclonal antibody to the G2-protein of Puumala virus. The antibody genes were inserted separately into plasmid transfer vector pIEI-4 such that the genes were under control of the baculovirus immediate early gene promoter, IEI. Trichoplusia ni (TN) cells were co-transfected with these constructs and a selection plasmid containing a neomycin-resistance gene. Cloned transformants expressing the IgG monoclonal antibody were identified by ELISA of transfected TN cell culture supernatants. TN cell lines were established from four selected clones, of which one was chosen for detailed analysis. Specificity of the insect cell-expressed human antibody was determined by ELISA with Puumala virus-infected cell lysates and by immune-precipitation of radiolabeled Puumala virus proteins. The expressed IgG retained the ability to neutralize Puumala virus in plaque-reduction neutralization assays. Using competitive polymerase chain reaction methods, multiple copies of integrated heavy- and light-chain antibody genes were detected in the insect cell genome. The transformed insect cells were stable and continuously expressed biologically active IgG. We conclude that this methodology provides an alternative eukaryotic source for the generation of human antibodies.
authors: Virology Division, United States Army Medical Research Institute of Infectious Diseases, Bldg. 1301, Fort Detrick, Frederick, MD 21702-5011, USA. mary.guttieri@det.amedd.army.mil
authors: M C
authors: MC
authors: Guttieri
authors: C
authors: C
authors: Bookwalter
authors: C
authors: C
authors: Schmaljohn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D018628:Gene Dosage; D015870:Gene Expression; D016678:Genome; D006476:Orthohantavirus / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007915:Lepidoptera / Q000235:genetics / Q000276:immunology / Q000821:virology; D051379:Mice; D014170:Transformation, Genetic; D014759:Viral Envelope Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014759:Viral Envelope Proteins