抗人肝细胞肝癌单克隆抗体的制备及鉴定

目的 制备高特异性和高亲和力的抗人肝细胞肝癌(HCC)的单克隆抗体(MAb),为肝癌的靶向诊断及治疗提供依据.方法 用人肝细胞肝癌细胞株HepG-2经"尾静脉.脾脏联合方法"免疫BALB/c小鼠.取其脾脏细胞与同源小鼠骨髓瘤细胞SP2/0-Ag14融合,经ABC免疫组化初筛抗体、有限稀释法亚克隆化、染色体分析、免疫组化鉴定抗体特异性、共聚焦显微镜扫描技术(LSCM)进行组织定位、ELISA分析鉴定抗体的亚型及荷人肝癌裸鼠牛物分布等进一步鉴定其生物学特性.结果 (1)获得一株分泌抗人肝细胞肝癌单克隆抗体的杂交瘤;该抗体与肝癌组织结合的特异性高达98.5%(67/68);(2)注射131I-MAb后瘤部位放射性分布有逐渐增加的趋势,血液、肝脏、肾脏和肺脏放射性有逐渐减低的趋势,72 h肿瘤/血液和肿瘤,肝脏比值分别为15.76±3.28和7.23±1.70.结论 成功制备抗人肝细胞肝癌的单克隆抗体,具有较好的肝癌特异性、靶向性,对原发性肝癌有潜在的诊断及治疗作用.

Establishment and identification of monoclonal antibody against human hepatoceilular carcinoma

Objective To establish and identify a new monoclonal antibody (MAb) against human hepatoceilular carcinoma (HCC) with high specificity and to provide evidence for targeted diagnosis and treatment of liver cancers. Methods Human HCC cell line HepG-2 was used to immunize BALB/c mice using the tail vein injection combined with intrasplenic injection. The sensitized splenic cells were fused with isogenic rat myeloma cells SP2/0-Ag14. The supernatant of the fused cells was detected by ABC immunohistochemistry (IHC) for primary antibody screening. The positive clones were subcloned using the limiting dilution method, and the chromosome of hybridoma was analyzed. The specificity of obtained MAb against HCC was identified by IHC. The epitope of the antigen was detected with laser confocal scanning microscope (LSCM). The subclass of the MAb was analyzed by enzyme linked immunosorbent assay (ELISA). The biodistribution of the MAb was detected in HCC-bearing nude mice to identify its biological characteristics. Results (1) One strain of hybridoma was obtained which produced antihuman HCC MAb with up to 98.5% (67/68) specificity in binding with liver cancer tissues. (2) After injection of 131I-MAb, radioactivity appeared increasingly stronger at the site of tumor and gradually weaker in the blood, liver, kidney and lungs. At 72 h, the ratio of radioactivity in tumor over blood and in tumor over liver was 15.76±3.28 and 7.23±1.70, respectively. Conclusions A novel MAb targeting well at HCC with high specificity is obtained. The MAb may be of potential use in diagnosis and treatment of primary HCC.