Zhong Xiufeng, Li Yongping, Ning Bo, Yan Jianhua, Li Yongqiang, Luo Guangwei, Lin Jianxian, Ying Fangwei, Feng Guanguang
Zhongshan Ophthalmic Center, Sun Yat-sen University, Eye Laboratory of Ministry of People's Health, Guangzhou 510060, China.
Zhonghua Yan Ke Za Zhi. 2002 Nov;38(11):691-4.
To prepare anti-human retinoblastoma (Rb) monoclonal antibody (McAb) by immunization with solid tumor cells and preliminarily study the characteristics of its antigen.
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.
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.
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 a possibility for a new unidentified gene to be concerned with the tumor formation of Rb.
通过实体瘤细胞免疫制备抗人视网膜母细胞瘤(Rb)单克隆抗体(McAb),并初步研究其抗原特性。
取摘除眼球获得的Rb实体瘤细胞,腹腔注射免疫3只Balb/C小鼠。分离其脾淋巴细胞与骨髓瘤细胞系SP2/0融合。以Rb实体瘤细胞、SO-Rb(50)、SO-Rb(70)等为抗原筛选抗人Rb McAb。通过免疫荧光、免疫组化及Western blot初步研究其抗原特性。
经显微操作仪反复克隆后,成功建立3株分泌抗人Rb McAb的杂交瘤细胞系。其中3C6杂交瘤细胞冻存复苏后连续传代2年能稳定分泌抗人Rb McAb,其亚类为IgG(1)。免疫荧光及免疫组化检测均显示,McAb 3C6的相应抗原在Rb肿瘤细胞膜及细胞质中特异性高表达,其他肿瘤组织及人正常眼组织均为阴性。Western blot分析初步表明,McAb 3C6能与Rb抗原的25 000蛋白条带结合。
通过实体瘤细胞免疫建立的抗人Rb McAb 3C6在Rb肿瘤细胞中特异性高表达,与其他肿瘤组织及正常眼球无交叉反应。该抗体所识别的抗原分子量约为25 000,进一步提示可能存在与Rb肿瘤发生相关的新未知基因。