Velikovsky C A, Cassataro J, Sanchez M, Fossati C A, Fainboim L, Spitz M
Instituto de Estudios de la Inmunidad Humoral (IDEHU), Facultad de Farmacia y Bioquímica, UBA Junin 956, 1113, Buenos Aires, Argentina.
J Immunol Methods. 2000 Oct 20;244(1-2):1-7. 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.
在脾脏内单次注射含有该蛋白质表达载体的DNA质粒48小时和25天后，产生了针对流产布鲁氏菌18 kDa蛋白的单克隆抗体（Mc. Abs.）。在融合前3天、5天和10天注射的脾脏也获得了杂交瘤。将注射了质粒DNA的小鼠脾脏细胞与NS - 0骨髓瘤细胞系在盐水中进行体细胞融合，产生了IgG和IgM同种型的Mc. Abs。IgG抗体为IgG2b和IgG1亚型。杂交瘤组织培养上清液在高达1/1200的稀释度下通过ELISA呈强阳性，并且在免疫印迹中产生强烈的特异性条带。所有这些抗体都识别天然重组蛋白（筛选抗原），其中一些还识别热变性的重组18 kDa蛋白。与标准免疫程序以及肌肉注射或基因枪DNA免疫相比，该技术可产生非常早期、节省时间且强效的Mc Abs。众所周知，为了产生特异性杂交瘤，免疫动物的脾脏细胞必须在最后一次加强免疫后不迟于5天进行融合。通过单次脾脏内免疫（SSI）在单次注射后25天产生特异性杂交瘤这一事实表明，编码p18蛋白的基因在脾脏中至少表达了20天。我们认为质粒DNA脾脏内免疫可以成为产生特异性杂交瘤的有用工具。这种免疫途径也有助于进一步了解通过裸DNA注射进行基因免疫时免疫反应的早期事件。
