抗人Atg5单克隆抗体的制备与鉴定

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／O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、westernB10t、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1E783H9，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×lO。，抗体亲和力常数为7．309×10^8mol／L；westernblot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。

Preparation and identification of monoclonal antibody against human Atg5

Objective To prepare and identify the monoclonal antibody （MAb） against human Atg5. Methods Splenocytes isolated from Balb/e mice which were immunized with recombinant protein Atg5 were fused with mouse myeloma cells SP2/0. After several rounds of screening and cloning, one stable strains of hybridoma was established. The specificity was evaluated with ELISA, immunohistoehemistry and Western blot, and the titer, immunoglobulin subtype and affinity constant of the mAbs were identified. Results One cell line of hybridoma named 1ETB3H9 was obtained, which was identified that the heavy and light chains of anti-human Atg5 MAb were IgG2b and ~, respectively. The mAb titer in ascetic fluid was 1：4. 096 x 105 and the affinity constant reached 7. 309 x 108mol/L, respectively. Western blot demonstrated that the antibody could specifically bind the natural protein of Atg5 in a cervical cancer cell line Hela lysat es. The cell immunohistoehemistry proved that the antibody eoulcl recognize the Atg5 protein expressed in the cytoplasm of Hela cells. Conclusion We have generated a monoelonal antibody, anti-human Atg5 protein with high specificity and affinity,which provides the basis for further study of the role and clinical application of Atg5 in disease with cervical cancer