title: Production of anti-dengue NS1 monoclonal antibodies by DNA immunization.
abstract: Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Bangkok 10700, Thailand.
authors.forename: Chunya
authors.initials: C
authors.lastname: Puttikhunt
authors.forename: Watchara
authors.initials: W
authors.lastname: Kasinrerk
authors.forename: Supa
authors.initials: S
authors.lastname: Srisa-ad
authors.forename: Thaneeya
authors.initials: T
authors.lastname: Duangchinda
authors.forename: Waraporn
authors.initials: W
authors.lastname: Silakate
authors.forename: Seangdeun
authors.initials: S
authors.lastname: Moonsom
authors.forename: Nopporn
authors.initials: N
authors.lastname: Sittisombut
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D019556:COS Cells; D003716:Dengue Virus / Q000276:immunology*; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection; D019444:Vaccines, DNA / Q000008:administration & dosage* / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; C079340:NS1 protein, Dengue virus type 2; D011994:Recombinant Proteins; D019444:Vaccines, DNA; D017361:Viral Nonstructural Proteins