抗人胰岛素样生长因子-1单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibody against human insulin-like growth factor-1

目的:制备稳定分泌抗人胰岛素样生长因子-1(hIGF-1)单克隆抗体(mAb)的杂交瘤细胞系,并对其分泌的mAb进行鉴定。方法:用纯化后的hIGF-1重组蛋白免疫Balb/c小鼠,利用杂交瘤细胞融合技术制备抗hIGF-1的mAb,用Western blot法鉴定mAb的特异性,用间接ELISA法检测mAb腹水效价及mAb的亲和力,杂交瘤细胞染色体分析,鉴定Ig亚类。结果:获得2株能稳定分泌抗hIGF-1的mAb杂交瘤细胞系,Western blot分析显示,该mAb能与具有生物学活性的成熟IGF-1蛋白结合。腹水mAb效价可达6×104。mAb相对亲和力为2.1×109/mol～7.8×109/mol。2株单抗属IgM亚类。染色体分析符合杂交瘤细胞特征。结论:成功制备出抗hIGF-1的2株mAb杂交瘤细胞,为进一步用于hIGF-1的临床检测和实验研究创造了条件。

Objective：To establish the specific hybridoma cell lines that stably secreted monoclonal antibody（mAb） against human insulin-like growth factor-1 and identificate its properties. Methods：Balb/c mice were immunized with purified recombinant human IGF-1 protein. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted human IGF-1 mAb were subcloned with limited dilution. The specificity of mAb was detected by Western blot. Indirect enzyme-linked immunosorbent assay（ELISA） was used to evaluate the affinity and titers of mAbs. The immunoglobulin （Ig） subtype of mAb was determined by test paper bar. Results：Two stably secreting anti-hIGF-1 mAb cell lines were obtained. Western blot analysis showed the mAb had specific combination with human IGF-1 which possessed biological activities. The titers of both mAb in ascites were 6 × 10^4 and their relative affinities were between 2.1 × 10^9/mol～7.8 × 10^9/mol. Two mAb cell lines can secret IgM subtype antibodies. Conclusion：Two hybridoma cell lines that secreted anti-hIGF-1 mAb were obtained,which will be useful for clinical application and fundamental basic research in hIGF-1.