Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes.
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.
Cantelli
Carina Pacheco
CP
Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil.
da Glória Martins Teixeira
Maria
M
Santos
Eneida Almeida
EA
da Silva
Haroldo Cid
HC
da Silva E Mouta
Sergio
S
Pimenta
Marcia Maria Araújo
MM
Vianna
Carlos Otávio Alves
CO
de Souza
Natália Plínio
NP
Batoreu
Nádia Maria
NM
Galler
Ricardo
R
de Moraes
Marcia Terezinha Baroni
MT
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
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D016899:Interferon-beta