EARLY DETECTION OF TAU ACETYLATION USING ULTRA-HIGH AFFINITY ANTIBODIES

使用超高亲和力抗体早期检测 TAU 乙酰化

• 批准号: 9227696
• 负责人: Yongku Peter Cho
• 金额: $ 7.47万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9227696
• 关键词: Acetylation; Address; Affinity; Alzheimer' s Disease; Antibodies; Antibody Affinity; Antibody Dissociations; Antibody Specificity; Antigens; Binding; Binding Sites; Biological; Brain; Cells; Chemicals; Chickens; Color; Communities; Deposition; Detection; Development; Directed Molecular Evolution; Disease Progression; Early Diagnosis; Engineering; Enhancing Antibodies; Ensure; Future; Generations; Genes; Homeostasis; Human; Hybridomas; Immune Sera; Immunoglobulin G; In Vitro; Investigation; Iowa; Label; Measurement; Measures; Mediating; Methods; Modification; Monitor; Monoclonal Antibodies; Nerve Degeneration; Neurodegenerative Disorders; Outcome; Passive Immunotherapy; Pathology; Patients; Post-Translational Protein Processing; Property; Reagent; Recombinant Antibody; Reproducibility; Resolution; Route; Scheme; Sensitivity and Specificity; Site; Specificity; Staging; Tauopathies; Testing; Tissue Sample; Transgenic Mice; Universities; Validation; antibody engineering; antibody libraries; base; clinically relevant; cross reactivity; high throughput screening; improved; insight; mouse model; mutant; neurotoxicity; novel; novel therapeutics; polyclonal antibody; prevent; process optimization; protein aminoacid sequence; research study; screening; tau Proteins; tau phosphorylation; tau-1; therapeutic development; tool

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.

项目摘要 tau的乙酰化是最近在由Tau介导的病理学的初始阶段中至关重要的机制。 最初的结果表明，tau乙酰化发生在疾病进展的早期，减少tau的周转率，并且 促进磷酸化tau的聚集。因此，早期检测到tau乙酰化并监测其 与tau磷酸化的协同作用是识别鉴定基础呼吸病的机制的有前途的途径。 但是，现有检测这种临界修饰的方法取决于多克隆抗体，这些抗体具有 特征性的结合特性不佳。在此提案中，我们假设提高了 检测乙酰化的tau将显着提高我们研究tau介导的初始阶段的能力 病理。但是，对特定蛋白质修饰位点产生高亲和力和特异性抗体是 具有挑战性的。这需要增强抗体亲和力，同时防止不必要的交叉反应性 相似的结合位点。即使已经开发了许多方法来筛选抗体序列 量化和工程师亲和力，基于交叉测量的优化抗体特异性的方法 反应性实际上是不存在的。为了解决这个问题，一种基于新颖的抗体筛选方法 将开发抗体特异性的定量度量。拟议的高通量筛查 方法将同时优化抗体特异性和亲和力。使用这种方法， 将产生针对特定于AD患者的关键乙酰化位点的高亲和力单克隆抗体。这 在此项目期间的具体目的是1）通过筛选确定乙酰化的tau结合抗体 重组抗体文库，2）通过定向进化来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氏病患者的组织样品验证抗体。 该项目的结果将是针对乙酰化tau的高质量单克隆抗体。如果成功， 该项目将使乙酰化tau早期检测，这对基本生物学产生了巨大影响 tau介导的神经退行性的研究和新的治疗发育。