抗人B7-H1单克隆抗体的制备和鉴定

Preparation and Identification of Anti-Human B7-H1 Monoclonal Antibody

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。

Objective： Preparation of anti-human B7-H1 monoclonal antibody with hybridoma technique and preliminary analysis of the application. Methods： The immunized mouse spleen cells and mouse myeloma cells were fused by routine method, indirect ELISA were used to screen the hybridoma. The positive clones were subcloned acquire hybridoma cells secreting anti-human B7-H1 monoclonal antibody. The amplified hybridoma cells were injected into abdominal cavity of mouse and the ascites were collected. The antibodies in the ascites were purified and titrated, and were indentified by immunohistochemical stain of lung cancer tissues. Results： One hybridoma cell line secreting anti-human B7-H1 monoclonal antibody was acquired. The subclass of the antibody was IgG1, the titer was 1×108, the concentration was 6.76 g / L after purification, and the antibody could combine with the B7-H1 which was on the surface of lung cancer tissues specifically. Conclusion： The anti-human B7-H1 monoclonal antibody was prepared successfully, which lay a foundation for later establishment of B7-H1 detecting kit