title: Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes.
abstract: Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.
authors.affiliation: Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil.
authors.forename: Carina Pacheco
authors.initials: CP
authors.lastname: Cantelli
authors.forename: Maria
authors.initials: M
authors.lastname: da Glória Martins Teixeira
authors.forename: Eneida Almeida
authors.initials: EA
authors.lastname: Santos
authors.forename: Haroldo Cid
authors.initials: HC
authors.lastname: da Silva
authors.forename: Sergio
authors.initials: S
authors.lastname: da Silva E Mouta
authors.forename: Marcia Maria Araújo
authors.initials: MM
authors.lastname: Pimenta
authors.forename: Carlos Otávio Alves
authors.initials: CO
authors.lastname: Vianna
authors.forename: Natália Plínio
authors.initials: NP
authors.lastname: de Souza
authors.forename: Nádia Maria
authors.initials: NM
authors.lastname: Batoreu
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
authors.forename: Marcia Terezinha Baroni
authors.initials: MT
authors.lastname: de Moraes
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology*; D016899:Interferon-beta / Q000276:immunology*; D051379:Mice; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D016899:Interferon-beta