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本研究旨在制备针对白血病干细胞表面标志蛋白IL1RAP(IL-1 receptor accessory protein)的特异性单克隆抗体并对其进行鉴定。用重组人IL1RAP作为抗原免疫BALB/c小鼠,通过常规杂交瘤技术将免疫后的小鼠脾细胞与SP2/0细胞经PEG融合,筛选阳性杂交瘤细胞株。分别应用ELISA和Western blot法对杂交瘤细胞上清中分泌抗体进行抗体类型、滴度和敏感性检测。分离人外周单个核细胞,检测所得抗体对细胞内源性抗原的特异性。结果表明,获得了8株可稳定分泌IL1RAP单克隆抗体的杂交瘤细胞株,分别为3H6E10、4B6A6、8G11B5、9E9F2、10D8A7、1C7H7、1D7G11和2D3D3;抗体类型鉴定为IgG1/κ型;分泌的单克隆抗体能特异性识别单个核细胞表达的IL1RAP。结论:本实验成功制备了可稳定分泌IL1RAP单克隆抗体的杂交瘤细胞及其特异性的IL1RAP单克隆抗体,为将来有效清除体内白血病干细胞提供了新途径。
The purpose of this study was to prepare and identify specific monoclonal antibodies against IL-1 receptor accessory protein. BALB / c mice were immunized with recombinant human IL1RAP as antigen, and the immunized mouse spleen cells and SP2 / 0 cells were fused with PEG by conventional hybridoma technique to screen positive hybridoma cell lines. Antibody type, titer and sensitivity of the secreted antibodies in the hybridoma cell supernatant were detected by ELISA and Western blot respectively. Peripheral mononuclear cells were isolated and the specificity of the resulting antibodies to the endogenous antigens of the cells was examined. The results showed that eight hybridoma cell lines secreting IL1RAP monoclonal antibody were obtained, which were 3H6E10, 4B6A6, 8G11B5, 9E9F2, 10D8A7, 1C7H7, 1D7G11 and 2D3D3, respectively. The type of antibody was identified as IgG1 / Clonal antibodies specifically recognize IL1RAP expressed by mononuclear cells. Conclusion: The hybridoma cells that can stably secrete IL1RAP monoclonal antibody and its specific IL1RAP monoclonal antibody were successfully prepared in this experiment, which provided a new way for the effective clearance of leukemic stem cells in vivo.