摘要
目的6B11是可模拟卵巢癌抗原的鼠源性抗独特型单克隆抗体,为了深入探讨其作为肿瘤疫苗的主动免疫机制,制备抗6B11的IgG型抗-抗独特型单克隆抗体并对其特性进行研究。方法以卵巢癌抗独特型抗体6B11作为免疫原,与载体钥孔槭血兰蛋白(KLH)偶联并辅以福氏佐剂,多次免疫同系Balb/c小鼠,通过杂交瘤技术将免疫脾细胞与骨髓瘤细胞Sp2/0融合;采用酶联免疫吸附实验(EL1SA)筛选杂交瘤细胞,经克隆化后建立稳定分泌鼠源性抗-抗独特型单克隆抗体的杂交瘤细胞株。秋水仙素法确认杂交瘤细胞染色体数目及形态,ELISA法鉴定抗体类型,采用非竞争酶免疫结合实验测定抗体亲和力,EL1SA、免疫组织化学方法检测抗-抗独特型单克隆抗体特异性结合抗原的性质。结果制备出一株能稳定分泌抗-抗独特型单克隆抗体的杂交瘤细胞株,命名为32F2。其染色体数目平均为103条,并有端着丝点和亚中部着丝点染色体,符合小鼠杂交瘤细胞特点。抗体类型为IgG1型,抗体亲和力约为2.3119×107L/mol,能够竞争抑制卵巢癌单克隆抗体COC166-9与原始抗原OC166-9的结合,免疫组织化学染色证实32F2鼠腹水与84.6%卵巢浆液性乳头状囊腺癌呈阳性染色,与COC166-9类似,表明32F2可与OC166-9抗原产生特异性结合。结论通过杂交瘤技术建立了分泌IgG1亚型的卵巢癌抗-抗独特型单克隆抗体的杂交瘤细胞株。对抗体的初步研究显示其能特异性结合初始抗原OC166-9,并能竞争抑制COC166-9与OC166-9的结合,属Ab1样Ab3,间接证实6B11为抗原内影像型抗体,具有模拟抗原作用。32F2的制备成功为进一步研究卵巢癌抗独特型疫苗6B11的作用机制打下了基础,并且该抗体具有潜在的临床治疗卵巢癌的价值。
Objective To generate and characterize a monoclonal anti - anti - idiotypic antibody for studying the mechanism of auto - actived immunotherapy of anti - idiotypic vaccine 6B11 against epithelial ovarian cancer which we had generated. Methods Monoclonal anti - idiotypic antibody 6Bll which bears the internal image of tumor associated antigen OC166- 9, coupled to KLH in Freud＇s adjuvant, was used as immunogen to induce the syngeneic mouse （Balb/c） to develop an anti - anti - idiotypic antibody response. Hybridoma cells were obtained by fusion of SP2/0 myeloma cells with immunized murine spleen cells. Positive clones were screened by indirect ELISA. MAb isotype was identified and its affinity was measured by noncompetitive enzyme immunoassay. To characterize the monoclonal antibody, ELISA, immunohistocbemistry were used to define its specific reactivity with the original antigen OC166 - 9. Results One hybridoma strain that could secrete anti - anti - idiotypic antibody （Ab3） was obtained by cell fusing, screening and cloning, then, name as 32F2. Its chromosomes are 103 at aver- age. IgG1 isotype was identified by ELISA. Its Kaff was approximately 2. 311 9 ×10^7 L/mol. It showed the ability of specific binding to the original antigen OC166 -9 from two respects.. Inhibiting the binding of Abl to OC166 -9; Immunohistochemistry cordirmed a positive staining of ovarian serous papillary carcinoma with Ab3 ascites. Conclusions We have generated one hybridoma strain which secretes anti - anti - idiotypic antibody of epithelial ovarian cancer. By characterizing the MAb, we confirmed it was Abl - like Ab3 and indirectly proved 6B11 beared the internal image of original antigen OC166 - 9.
出处
《中国妇产科临床杂志》
2007年第6期445-449,I0002,共6页
Chinese Journal of Clinical Obstetrics and Gynecology
基金
国家自然科学基金项目(30471949)资助
关键词
卵巢癌
抗独特型抗体
抗-抗独特型抗体
Ovarian cancer
Anti - idiotypic antibody
Anti - anti - idiotypic antibody
