Generation of Monoclonal Antibodies Against Human Recombinant Interferon Beta Using Genetic Immunization with Simultaneous Expression of IgM and IgG Isotypes

摘要

Monoclonal antibodies (MAbs) against human recombinant interferon β (hrIFNβ) 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 100μg of either pcDNA 3.1hrIFNβ or pZeoSV2IFNβ containing the entire human interferon β 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 pZeoSV2IFNβ were positive to anti-hrIFNβ. 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 hrIFNβ. Western blot analysis was performed and only one recognized the 18kDa isoform (non-glycosylated) of hrIFNβ. 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.