PURIFICATION OF A FIBRINOLYSIS ENHANCING PROTEIN

纤溶增强蛋白的纯化

• 批准号: 3502128
• 负责人: MARK X TRISCOTT
• 金额: $ 5万
• 依托单位: AMPLISTAR, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3502128
• 关键词: antibody; bioassay; blood proteins; electrophoresis; enzyme activity; enzyme linked immunosorbent assay; fibrinolysis; fibrinolytic agents; heparin; high performance liquid chromatography; human tissue; immunoaffinity chromatography; immunologic assay /test; ion exchange chromatography; laboratory rabbit; method development; monoclonal antibody; plasmin; protein purification; protein structure function

During the development of our fibrinolytic assay system we noted an enhancement of fibrinolytic activity in plasma which could not be accounted for by any known fibrinolytic factor. Using heparin affinity chromatography, with a 0.5M NaCl elution and an immuno affinity column we have been able to partially purify this factor. The immuno affinity column uses immobilized MCab-3, a monoclonal antibody against a plasma eluate from a heparin affinity column. We have demonstrated the enhancing activity of the protein in the enzyme linked fibrinolytic assay (ELFA), a clot lysis assay with added t-PA, and a bio immunoassay using a transferable solid phase enzyme labelled fibrin. Using the bio immunoassay we have estimated plasma levels to be approximately 4.5 units. Plasma protein levels for the cofactor appear to be less than 50ug/ml. HPLC gel permeation chromatography reveals a MCab-3 binding protein of 78KD. The protein could not be activated in the presence of high concentrations of extrinsic fibrinolytic activators, precluding the possibility of plasminogen being responsible for the activity. The protein did not activate plasminogen and had no measurable fibrinolytic activity. Immunodiffusion analysis of the intact protein revealed non identity with vitronectin or histidine rich glycoprotein, other heparin binding plasma proteins implicated in fibrinolysis modulation. We propose to purify the protein to homogeneity and positively identify it by N-terminal sequence analysis. We propose raising a polyclonal antibody against the protein to use in the development of immunoassays for the measurement of the protein in plasma.

在我们的纤溶测定系统的开发过程中，我们注意到 血浆中纤维蛋白溶解活性的增强，但无法解释 对于任何已知的纤溶因子。 使用肝素亲和力 层析，用 0.5M NaCl 洗脱和免疫亲和柱 已经能够部分纯化这个因子。 免疫亲和柱 使用固定化 MCab-3，这是一种针对血浆洗脱液的单克隆抗体 肝素亲和柱。 我们已经证明了增强活性 酶联纤溶测定 (ELFA) 中的蛋白质，即凝块溶解 添加 t-PA 的测定，以及使用可转移固体的生物免疫测定 相酶标记的纤维蛋白。 使用我们估计的生物免疫分析 血浆水平约为 4.5 个单位。 血浆蛋白水平为 辅因子似乎低于 50ug/ml。 HPLC凝胶渗透 色谱法显示 78KD 的 MCab-3 结合蛋白。 该蛋白质可以 在高浓度的外源性物质存在下不会被激活 纤维蛋白溶解激活剂，排除纤溶酶原的可能性 负责该活动。 该蛋白质不激活纤溶酶原并且 没有可测量的纤溶活性。 免疫扩散分析 完整蛋白质与玻连蛋白或富含组氨酸不同 糖蛋白，其他肝素结合血浆蛋白涉及 纤溶调节。 我们建议将蛋白质纯化至均质 并通过N端序列分析对其进行肯定鉴定。 我们建议 产生针对该蛋白质的多克隆抗体以用于开发 用于测量血浆中蛋白质的免疫测定法。