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

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein fr om Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridom as 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 Me. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hy bridoma tissue culture supernatants were strongly positive by ELISA at dilu tions of up to 1/1200 and produced intense specific bands in immunoblotting . All these antibodies recognized the native recombinant protein (the scree ning antigen) and some of them also recognized the heat-denatured recombina nt 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gent: gun DNA immunizations, this technique re sults in very early, time saving, strong Me Abs. It is common knowledge tha t in order to generate specific hybridomas; spleen cells from immunized ani mals have to be fused no later than 5 days after the last boost. The fact t hat through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene c oding 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 to ol for the production of specific hybridomas. This route of immunization co uld also be helpful in the further understanding of early events of the imm une response to genetic immunization by naked DNA injection. (C) 2000 Elsev ier Science B.V. All rights reserved.