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Preparation and identification of monoclonal antibody against human thrombomodulin
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- Author:
- No author available
- Journal Title:
- ACTA PHYSIOLOGICA SINICA
- Issue:
- 4
- DOI:
- 10.3321/j.issn:0371-0874.2006.04.015
- Key Word:
- 血栓调节蛋白;表达;单克隆抗体
Abstract： 为了制备特异性抗人血栓调节蛋白(human thrombomodulin,bTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含bTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株.将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA(cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水.用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定.我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgG1,McAb腹水效价为1×10-6,腹水抗体含量为20 mg/ml.NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的bTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础.
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