EARLY DETECTION OF TAU ACETYLATION USING ULTRA-HIGH AFFINITY ANTIBODIES

项目摘要 Tau乙酰化是新近发现的一种机制，在tau介导的病理过程的初始阶段起关键作用。 初步结果表明，tau乙酰化发生在疾病进展的早期，减少tau的周转， 促进磷酸化tau的聚集。因此，早期检测tau的乙酰化并监测其 与tau磷酸化的协同作用是确定tau病潜在机制的一条很有前途的途径。 然而，现有的检测这种关键修饰的方法依赖于多克隆抗体，这些抗体已经 不能很好地描述绑定特性。在这项提议中，我们假设增强对 检测乙酰化tau将极大地提高我们研究tau介导的初始阶段的能力 病理学。然而，产生针对特定蛋白修饰位点的高亲和力和特异性抗体是 很有挑战性。这需要增强抗体亲和力，同时防止不必要的交叉反应 相似的结合位点。尽管已经开发了许多方法来筛选抗体序列以 量化和设计亲和力，基于交叉测量优化抗体特异性的方法 反应性实际上是不存在的。为了解决这一问题，提出了一种新的抗体筛选方法 将开发抗体特异性的定量测量方法。建议的高通量筛选 该方法将能够同时优化抗体的特异性和亲和力。使用这种方法，超 将产生针对AD患者的关键乙酰化位点的高亲和力单抗。这个 本项目期间的具体目标是：1)通过筛选鉴定乙酰化tau结合抗体 重组抗体库，2)通过定向进化优化抗体的特异性和亲和力， 3)使用小鼠模型和阿尔茨海默病患者的组织样本对抗体进行验证。 该项目的成果将是针对乙酰化tau的高质量单抗。如果成功， 该项目将能够及早检测乙酰化tau，这对基础生物学有巨大影响。 Tau介导的神经变性的研究和新的治疗进展。

Project Summary Acetylation of tau is a recently identified mechanism critical in the initial stages of pathology mediated by tau. Initial results show that tau acetylation occurs early on in disease progression, reduces tau turnover, and promotes aggregation of phosphorylated tau. Therefore, early detection of tau acetylation and monitoring its synergy with tau phosphorylation is a promising route to identifying the mechanism underlying tauopathies. However, existing methods to detect this critical modification have relied on polyclonal antibodies, which have poorly characterized binding properties. In this proposal, we hypothesize that enhancing the sensitivity of detecting acetylated tau would dramatically improve our ability to study the initial stages of tau mediated pathology. However, yielding high affinity and specificity antibodies against specific protein modification sites is challenging. This requires enhancing antibody affinity while preventing unwanted cross reactivity towards similar binding sites. Even though numerous methods have been developed to screen antibody sequences to quantify and engineer affinity, methods to optimize antibody specificity based on measurement of cross reactivity are virtually non-existent. To address this problem, a novel antibody screening approach based on quantitative measure of antibody specificity will be developed. The proposed high-throughput screening approach will enable simultaneous optimization of antibody specificity and affinity. Using this approach, ultra- high affinity monoclonal antibodies against key acetylation sites specific to AD patients will be generated. The specific aims during this project period are 1) Identifying acetylated tau binding antibodies by screening recombinant antibody libraries, 2) optimizing specificity and affinity of the antibodies through directed evolution, and 3) validation of the antibodies using tissue samples from mouse models and Alzheimer's disease patients. The outcomes of this project will be high quality monoclonal antibodies targeting acetylated tau. If successful, the project will enable early detection of acetylated tau, which has enormous impact on basic biological investigations and novel therapeutic development in tau mediated neurodegeneration.