Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies: Persistent expression of DNA

• Published in: Journal of immunological methods 2000-10, Vol.244 (1), p.1-7
• Main Authors: Velikovsky, Carlos A., Cassataro, Juliana, Sanchez, Mercedes, Fossati, Carlos A., Fainboim, Leonardo, Spitz, Moises
• Format: Article
• Language: English
• Subjects: Animal cells; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Specificity; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; Bacteriology; Biological and medical sciences; Biotechnology; Brucella abortus; Brucella abortus - genetics; Brucella abortus - immunology; Cell Fusion; Cell hybridization, cell fusion, hybridomas; DNA - administration & dosage; DNA - genetics; DNA - immunology; Eukaryotic cell cultures; Fundamental and applied biological sciences. Psychology; Hybridomas; Immunization Schedule; Immunoglobulin Isotypes - biosynthesis; Immunoglobulin Isotypes - genetics; Immunoglobulin Isotypes - immunology; Intrasplenic immunization; Lipoproteins; Methods. Procedures. Technologies; Mice; Microbiology; Monoclonal antibodies; Multiple Myeloma - immunology; Multiple Myeloma - metabolism; Naked DNA; Plasmids - genetics; Spleen - immunology; Spleen - metabolism; Tumor Cells, Cultured; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies; Vaccines, DNA - administration & dosage; Vaccines, DNA - genetics; Vaccines, DNA - immunology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00244-1

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.