视网膜母细胞瘤组织直接免疫制备抗人视网膜母细胞瘤单克隆抗体及其抗原特性的实验研究

Preparation of anti-human retinoblastoma monoclonal antibody by solid tumor cell immunization and study on characteristics of its antigen

目的 研究新鲜视网膜母细胞瘤 (retinoblastoma ,Rb)细胞制备的抗人Rb单克隆抗体(monoclonalantibody ,McAb)的抗原特性。方法 利用手术中获得的新鲜Rb组织细胞制备成细胞悬液 ,并将其注射入 3只BALB C小鼠腹腔中 ,免疫小鼠。取其脾淋巴细胞与SP2 / 0骨髓瘤细胞融合 ,以新鲜Rb细胞及Rb细胞株等为抗原筛选特异性抗体。用免疫荧光、免疫组化及免疫印迹法对其相应抗原的性质进行检测 ,观察McAb相应抗原蛋白在Rb组织中的表达。结果 经反复单细胞克隆化操作及抗体特异性筛选 ,共获得 3株能持续、稳定分泌抗人RbMcAb的杂交瘤细胞系。其中 3C6株杂交瘤细胞在体外经≥ 2年反复冻存 ,传代培养 ,仍能稳定分泌特异性强、效价高的抗体 ,为IgG1亚类。免疫荧光及免疫组化检测结果显示 ,McAb 3C6相应抗原蛋白在Rb组织中呈强阳性表达 ,而在其他肿瘤及正常组织中无表达。免疫印迹法检测表明 ,McAb 3C6相应抗原蛋白相对分子质量约 2 5 0 0 0。结论采用新鲜的实体瘤细胞制备的抗人RbMcAb 3C6在Rb组织中呈高度特异性表达 ,与其他肿瘤及正常眼组织无交叉反应 ;其所针对的抗原蛋白相对分子质量约为 2 5 0 0 0 。

Objective To prepare anti human retinoblastoma (Rb) monoclonal antibody (McAb) by immunization with solid tumor cells and preliminarily study the characteristics of its antigen. Methods Three Balb/C mice were immunized by intraperitoneal injection of Rb solid tumor cells acquired from enucleation. Spleen lymphocytes were separated from them and fused with myeloma cell line SP2/0. The anti human Rb McAb was selected by Rb solid tumor cell, SO Rb 50 , SO Rb 70 , etc as antigens. The characteristics of its antigen were preliminarily studied by immunofluorescence,immunohistochemistry and Western blot. Results After repeatedly cloning them with micro manipulation equipment, we successfully established 3 hybridoma cell lines secreting anti human Rb McAb. Of all, 3C6 hybridoma cells could steadily secrete anti human Rb McAb after they were frozen, resuscitated and passaged repeatedly for 2 years. Its subgroup was IgG 1. Both immunofluorescence and immunohistochemistry examinations demonstrated that the corresponding antigen of McAb 3C6 was specifically and highly expressed in Rb tumor cell membrane and cytoplasm. The other tumor tissues and human normal eye tissues were negative. Western blot analysis preliminarily demonstrated that McAb 3C6 could bind 25 000 protein band of Rb antigen. Conclusions The anti human Rb McAb 3C6 established by immunization of solid tumor cells is specifically and highly expressed in Rb tumor cells and has no cross response with other tumor tissues and normal eyeballs. The antigen molecular weight bounded to this antibody is 25 000 or so, which further shows that it is apossibility for a new unidentified gene to be concerned with the tumor formation of Rb.