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目的制备抗人Atg5单克隆抗体,并对其生物学特性进行鉴定。方法以人Atg5重组蛋白作为免疫原免疫Balb/c小鼠,取其脾细胞与SP2/0细胞融合,经多次筛选及克隆化,建立可稳定分泌抗人Atg5单克隆抗体的杂交瘤细胞株。用ELISA、Western Blot、免疫组化鉴定单克隆抗体的特性,并测定其效价、亚型及亲和力常数。结果成功筛选到一株能稳定分泌抗人Atg5单抗的细胞株1E7B3H9,亚型鉴定重链为IgG2b,轻链为kappa链。ELISA法测定腹水抗体效价为1:4.096×105,抗体亲和力常数为7.309×108 mol/L;Western blot显示此单抗能特异性识别人子宫颈癌Hela细胞系中的天然人Atg5蛋白;细胞免疫组化进一步显示Atg5表达在细胞质中。结论成功制备了抗人Atg5单克隆抗体,为进一步研究其与宫颈癌关系及在临床上的应用奠定了基础。
Objective To prepare anti-human Atg5 monoclonal antibody and identify its biological characteristics. Methods Balb / c mice were immunized with human Atg5 recombinant protein. The spleen cells were fused with SP2 / 0 cells. After screening and cloning for several times, a hybridoma cell line stably secreting anti-human Atg5 monoclonal antibody was established . The characteristics of the monoclonal antibodies were identified by ELISA, Western Blot and immunohistochemistry. The titer, subtype and affinity constants were also determined. Results A stable cell line secreting anti-human Atg5 McAb 1E7B3H9 was successfully screened. The subtype was identified as IgG2b and the light chain was kappa chain. The titer of ascites antibody was 1: 4.096 × 105 by ELISA and the antibody affinity constant was 7.309 × 108 mol / L. Western blot showed that this monoclonal antibody could specifically recognize native human Atg5 protein in human cervical cancer Hela cell line. Immunohistochemistry further showed that Atg5 was expressed in the cytoplasm. Conclusion The anti-human Atg5 monoclonal antibody was successfully prepared, which laid the foundation for further study on its relationship with cervical cancer and its clinical application.