卵巢癌抗抗独特型单克隆抗体3D12的制备及初步研究

目的:制备卵巢癌抗抗独特型单克隆抗体并进行初步鉴定,为深入研究卵巢癌疫苗的主动免疫机制奠定基础.方法:以卵巢癌抗独特型单克隆抗体6B11作为免疫原,采用杂交瘤技术制备抗抗独特型单克隆抗体3D12.经酶联免疫吸附实验(ELISA)、免疫组织化学、流式细胞学、免疫印迹杂交等检测其特异性结合抗原的性质;竞争抑制实验鉴定其特异性结合的抗原表位.结果:获得1株稳定分泌3D12的杂交瘤细胞,所分泌的3D12能够特异性结合6B11及卵巢癌初始抗原OC166-9,并能竞争抑制COC166-9(抗OC166-9的单克隆抗体Ab1)与OC166-9的结合.免疫印迹杂交显示,3D12与COC166-9免疫沉淀的蛋白的相对分子质量相同.该抗体为IgM,亲和力约为8.34×10⁷ L/mol.结论:卵巢癌抗抗独特型单克隆抗体3D12属Ab1样Ab3,间接证实6B11为抗原内影像型抗独特型抗体.

Preparation and characterization of monoclonal anti-anti-idiotypic antibody (3D12) of epithelial ovarian carcinoma

OBJECTIVE: To generate and characterize the anti-anti-idiotypic monoclonal antibody (MAb) of epithelial ovarian carcinoma (EOC)ìwith the aim of further investigating the mechanism of active immunotherapy of tumor vaccine. METHODS: Using anti-idiotypic MAb (6B11)of EOC as immunogen, the anti-anti-idiotypic MAb 3D12 was generated by hybridoma technology. To characterize the 3D12, ELISA, immunohistochemistry, flow cytometry and western blot were used to define its specific reactivity with both 6B11 and the original EOC antigen OC166-9. RESULTS: One hybridoma cell secreting 3D12 was obtained. The 3D12 showed the ability of specific binding to 6B11 and OC166-9, and of inhibiting the binding of COC166-9 (the MAb against OC166-9) to OC166-9. A positive staining of serous papillary EOC and the specific binding of SKOV3 cell line with 3D12 supernatant were also demonstrated. Western blot showed 3D12 and COC166-9 could immunoprecipitate the same protein. The 3D12 was IgM, with the affinity Kaff approximately 8.34x10(7) L/mol. CONCLUSION: The anti-anti-idiotypic MAb 3D12 is confirmed as Ab1-like Ab3, which indirectly proves that 6B11 bears the internal image of OC166-9.