A NEW PROTEIN IN ALZHEIMER'S DISEASE

在过去一个时期，在以下方面取得了重大进展： 了解阿尔茨海默病大脑中Alz-50抗原的性质， 并使用免疫亲和层析与IgG 1类别转换变体 的Alz-50，分离成对螺旋丝（PHF）的新方法 开发了 通过这种技术提纯的材料已被用于 提出了两个新的单克隆抗体系列， 已经开发了PHF的免疫亲和纯化。 在这 完全修改后的应用程序，我们建议进一步表征 纯化的PHF，使用生物化学、免疫印迹和HPLC技术， 和结构（圆二色性，傅立叶变换红外 光谱、X射线衍射）分析，以及电子 显微镜（特定目标1）。 将尝试界定这一作用 特定的多肽和功能基团的组装配对 螺旋状细丝 一种蛋白激酶活性存在于 纯化的PHF制剂将在特定目标中进一步表征 2，特别是在底物特异性方面。 进一步 还将对新的单克隆抗体进行表征， 出去 两个特别有趣的新单克隆抗体已经被 在项目的最后一个阶段。 TG 3是一种IgM， 是形成固定的免疫细胞化学研究中的上级试剂， 石蜡包埋的人体尸检组织 前期工作已经 不能证明该抗体与磷酸化的 tau，尽管与成对的螺旋丝有反应性。 下 具体目标3，PHF中TG 3表位的性质的研究将是 执行。 MC 16是一种IgG 2a，其在正常和非正常细胞中染色细胞核。 AD脑，但也与缠结和神经炎元素反应， AD大脑中的淀粉样蛋白 通过免疫印迹，主要反应蛋白 似乎是一个3 kDa的蛋白质，并且该抗体没有反应性 与磷酸化的tau蛋白结合 在特定目标4中，该蛋白质将被 从人脑中纯化并测序以确定其身份。 给定 大量不同的PHF相关表位，现在可以 检查，尝试将在具体目标5使用免疫印迹 和ELISA技术来分期这些表位的出现， 与AD病理学发展的关系，使用神经病理学 布拉克的分期系统。

Over the last period, significant progress has been made in understanding the nature of the Alz-50 antigen in the Alzheimer brain, and using immunoaffinity chromatography with an IgG1 class switch variant of Alz-50, a new method for isolation of paired helical filaments (PHF) has been developed. Material purified by this technique has been used to raise two new series of monoclonal antibodies, and a more efficient immunoaffinity purification of PHF has been developed. In this completely revised application, we propose to further characterize the purified PHF, using both biochemical, immunoblotting and HPLC techniques, and structural (circular dichroism, Fourier-transformed infrared spectroscopy, X-ray diffraction) analysis, together with electron microscopy (Specific Aim 1). Attempts will be made to define the role of specific polypeptides and functional groups on the assembly of paired helical filaments. A protein kinase activity present in the highly purified PHF preparations will be further characterized in Specific Aim 2, especially in terms of substrate specificity. Further characterization of the new monoclonal antibodies will also be carried out. Two particularly interesting new monoclonal antibodies have been developed over the last project period. TG3 is an IgM which appears to be superior reagent in immunocytochemical studies of formation fixed and paraffin embedded human autopsy tissues. Preliminary work has been unable to demonstrate any reactivity of this antibody with phosphorylated tau, although there is reactivity with paired helical filaments. Under Specific Aim 3, studies of the nature of the TG3 epitope in PHF will be performed. MC16 is an IgG2a which stains nuclei in both the normal and AD brain, but also reacts with tangles and with neuritic elements and amyloid in the AD brain. By immunoblotting, the major reactive protein appears to be a 3kDa protein, and there is no reactivity of this antibody with phosphorylated tau. In Specific Aim 4, this protein will be purified from human brain and sequenced to determine its identity. Given the large number of different PHF-related epitopes which can now be examined, attempts will be made in Specific Aim 5 to use immunoblotting and ELISA techniques to stage the appearance of these epitopes in relation to the development of AD pathology, using the neuropathologic staging system of Braak.