THROMBUS DETECTION USING FIBRIN-SPECIFIC ANTIBODIES

目前的体内血栓检测方法要么缺乏 特异性或造成额外的健康风险。 借助 分子免疫学和合成肽化学， 以制备临床上有用的诊断抗体， 对人纤维蛋白（血栓中的结构蛋白）的特异性， 通过以下方式为体内血栓检测提供足够的灵敏度： 伽玛射线照相术 我们的战略将强调 纤维蛋白及其可溶性前体纤维蛋白原之间的关系， 选择纤维蛋白特异性抗体的方法 合成肽 将被设计成模拟纤维蛋白独特的表位，并用于引发 在存在下结合人纤维蛋白的抗体 纤维蛋白原。 这种方法在上一次赠款期间得到了验证 当我们使用对应于N的合成肽时 人纤维蛋白β-链的末端， 在存在下与纤维蛋白单体结合的抗体 纤维蛋白原。 一种单克隆抗体的体内特异性 （64 C5）在使用鸡、兔和 狗模特 在这里提出的研究中，我们专注于设计 并测试各种纤维蛋白样肽， 凝血酶切割位点和因子XIIIa交联位点，和 描述使用纤维蛋白的改进筛选策略的计划 微片段而不是纤维蛋白单体作为抗原。 我们的具体 目的包括在体内测试抗体5D 7作为γ-干扰素， 血栓造影剂，用于检测实验性血栓; 与凝块紧密结合的单克隆抗体的制备 纤维蛋白;相关交联抗原的合成和精制 连接到γ链交联位点;并且结构和 α链交联位点的免疫化学研究， 以鉴定新的纤维蛋白独特表位。 三个具体好处 将从这些实验中产生：首先，他们将提供一个 纤维蛋白上独特表位的详细免疫化学分析 分子。 他们还将提出一般方法， 将任何蛋白质与其前体区分开来。 第二，他们将 提供了可以在体内标记血栓的试剂。 第三，他们会奠定 一种敏感而特异的非侵入性 可以建立对危及生命血栓栓塞的测试。

Current methods for in vivo thrombus detection either lack specificity or pose additional health risks. With the tools of molecular immunology and syntheitic peptide chemistry, we plan to prepare a clinically useful, diagnostic antibody that possesses specificity for human fibrin (the structural protein in thrombi) and provides sufficient sensitivity for in vivo thrombus detection by gamma scintigraphy. Our strategy will emphasize the differences between fibrin and its soluble precursor, fibrinogen, and will focus on ways to select fibrinspecific antibodies. Synthetic peptides will be designed to mimic fibrinunique epitopes and used to elicit antibodies that will bind human fibrin in the presence of fibrinogen. This approach was validated during the previous grant period when we used a synthetic peptide corresponding to the N terminus of the human fibrin beta-chain to elicit monoclonal antibodies that bound to fibrin monomer in the presence of fibrinogen. The in vivo specificity of one monoclonal antibody (64C5) was confirmed in experiments using chicken, rabbit and dog models. In the research proposed here, we focus on the design and testing of various fibrin-like peptides corresponding to thrombin cleavage sites and Factor XIIIa cross-link sites, and describe plans for an improved screening strategy that uses fibrin miniclots rather than fibrin monomers as antigens. Our specific aims include the in vivo testing of antibody 5D7 as a gamma scintigraphic agent for detecting experimental thrombi; the preparation of monoclonal antibodies that avidly bind to clotted fibrin; the synthesis and refinement of cross-link antigens related to gamma-chain cross-link sites; and the structural and immunochemical study of the alpha-chain cross-link site in order to identify a new fibrin-unique epitope. Three specific benefits will result from these experiments: First, they will provide a detailed immunochemical analysis of unique epitopes on the fibrin molecule. They will also suggest general methods for differentiating any protein from its precursor. Second, they will provide reagents that can tag thrombi in vivo. Third, they will lay the foundation upon which a sensitive and specific noninvasive test for life-threatening thromboembolism can be built.