| 摘要: |
| 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。 |
| 关键词: 合成多肽 单克隆抗体 信号调节蛋白α THP-1 |
| DOI：10.3724/SP.J.1008.2010.0900 |
| 投稿时间：2010-03-18修订日期：2010-06-09 |
| 基金项目: |
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| Preparation and preliminary application of a novel monoclonal antibody against human SIRPα |
| TANG Liang, YAN He-xin, LIU Qiong, TANG Shan-hua,,WANG Hong-yang |
| (International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai 200438, China) |
| Abstract: |
| To prepare a monoclonal antibody against human SIRPα using synthetic peptide, and to use it for immune test to assess its efficacy. Methods The synthetic peptide of SIRPα was linked with KLH and the product was used as antigen for immunization of BALB/c mice. The mAb anti-SIRPα was obtained by hybridoma technique. The produced mAb was used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Cytokines secreted by PMA-treated THP-1 cells were tested by antibody arrays after exposure to the obtained mAb, and the levels of TNF-α and IL-6 were assayed by ELISA. Results The mAb secreting hybridoma clone was successfully obtained and it had a satisfactory efficacy when used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Compared with negative control and isotype control, the prepared mAb can stimulate TNF-α and IL-6 secretion in PMA-treated THP-1 cells. Conclusion We have successfully prepared the mAb against SIRPα using synthetic peptide. |
| Key words: synthetic peptide monoclonal antibodies signal regulatory protein alpha THP-1 |
