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目的以合成的多肽制备人信号调节蛋白α(SIRPα)的单克隆抗体(mAb),并初步应用于免疫学检测,评价其应用效果。方法以合成多肽与载体KLH交联的偶联物为免疫原,免疫BALB/c小鼠,应用细胞融合技术建立能稳定分泌抗人SIRPα的杂交瘤细胞株。采用分泌的mAb进行流式细胞术、蛋白质印迹及免疫组化检测,观察检测结果;采用分泌的mAb刺激经豆蔻佛波醇乙酯(PMA)处理的THP-1细胞,抗体芯片检测分泌细胞因子水平的变化,ELISA法验证其中TNF-α、IL-6的水平变化。结果成功获取可分泌抗人SIRPα的杂交瘤细胞,分泌的mAb应用于流式细胞术、蛋白质印迹及免疫组化检测,取得较好的效果;与阴性对照组和同型抗体对照组相比,制备的mAb能促进经PMA处理的THP-1细胞分泌TNF-α、IL-6等细胞因子(P<0.01)。结论利用合成多肽成功制备了高特异性抗人SIRPα的单克隆抗体。
OBJECTIVE: To prepare monoclonal antibody (mAb) of human signal regulatory protein alpha (SIRPα) using synthetic peptides and to preliminary apply it to immunological detection to evaluate its application effect. Methods BALB / c mice were immunized with the conjugate of KLHH. The hybridoma cell lines stably secreting anti - human SIRPα were established by cell fusion technique. The secreted mAbs were used for flow cytometry, Western blotting and immunohistochemistry to observe the test results. THP-1 cells stimulated with PMA were stimulated with secreted mAbs, and antibody chip was used to detect the secretion of cytokines The level of TNF-αand IL-6 in serum were detected by ELISA. Results The hybridoma cells secreting anti-human SIRPα were successfully obtained. The secreted mAbs were used in flow cytometry, Western blot and immunohistochemistry to obtain better results. Compared with the negative control group and the same antibody group, MAb could promote the secretion of TNF-α, IL-6 and other cytokines by PMA-treated THP-1 cells (P <0.01). Conclusion The monoclonal antibodies against human SIRPα were successfully prepared using synthetic peptides.