Analytical validation of a biochemical test for alpha-synuclein aggregates in biological fluids for the diagnosis of Parkinson's Disease

项目总结 错误折叠的蛋白质聚集体是有毒的超分子蛋白质集合体，它自身 在宿主体内复制，对周围组织造成损害。这些蛋白质聚集体中有几个是 与神经退行性疾病有很强的相关性，例如普恩病毒疾病中的普恩蛋白， 淀粉样蛋白β和tau在阿尔茨海默病和肌萎缩侧索硬化症中的表达 额颞叶痴呆和α-突触核蛋白(αS)在帕金森病(PD)中的作用，痴呆与 路易小体(DLB)和多发性系统性萎缩(MSA)。IN背后的分子机制 这些聚集体的体内形成及其对神经组织的影响仍然难以捉摸，但它们的 令人信服的暗示，疾病过程的早期驱动力确定了它们是潜在的有用的 疾病生物标志物和治疗靶点。该提案的主要目标是实现可靠的性能 蛋白质错折叠循环扩增法检测αS的分析验证 共核病患者脑脊液和血浆中的聚集物。PMCA Platform利用错误折叠的蛋白质聚集体的内在自我复制特性来循环放大 微量的αS聚集在生物样品中。该分析在96孔板中进行， 并且随着时间的推移，使用荧光淀粉样特异性染料硫黄素T来监测扩增。 方法开发的重点是稳健性，以支持未来在受控实验室中的使用 高样品吞吐量和对分析准确性和重现性的严格要求。分析性 验证将根据FDA和临床实验室标准协会(CLSI)进行 建议，使用临床阳性患者样本和添加了 人工生成的聚集体来评估方法的准确性、精密度、灵敏度、特异度、 可报告的范围和其他关键的化验特性。第一阶段的总体目标是 最终确定标准操作程序和协议，并执行以下分析验证 定性/半定量αS-PMCA法检测脑脊液和血浆中αS寡聚体 作为帕金森病和相关联核病的诊断生物标记物。第二阶段的总体目标 二是修改脑脊液和血浆中αS低聚物的定量/动力学表征方法， 并探索扩大使用范围以包括以下适应症：易感性/风险生物标记物 在临床症状出现之前确定有可能发展为联体核病的患者；a 区分不同类型联体核病的诊断生物标记物；监测 用于评估疾病进展或治疗效果的生物标记物；以及具有 描述疾病类型和严重程度的能力。

PROJECT SUMMARY Misfolded protein aggregates are toxic supramolecular proteinaceous conglomerates that self- replicate in the host, causing damage to surrounding tissue. Several of these protein aggregates are strongly associated with neurodegenerative diseases, such as the prion protein in prion diseases, Amyloid β and tau in Alzheimer’s (AD), TDP-34 in amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD), and α-synuclein (αS) in Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and Multiple Systemic Atrophy (MSA). The molecular mechanisms behind the in vivo formation of these aggregates and their effect on neural tissue remain elusive, but their compelling implication as early drivers of the disease processes identifies them as potentially useful disease biomarkers and therapeutic targets. The primary goal of this proposal is to perform robust analytical validation of a Protein Misfolding Cyclic Amplification (PMCA) assay for detection of αS aggregates in cerebrospinal fluid (CSF) and plasma of patients with synucleinopathies. The PMCA platform uses the intrinsic self-replicating nature of misfolded protein aggregates to cyclically amplify minute quantities of αS aggregates in biological samples. The assay is performed in a 96-well plate, and the amplification is monitored over time using the fluorescent amyloid-specific dye, ThioflavinT. Method development is focused on robustness to support future use in a controlled laboratory with high sample throughput and strict requirements for assay accuracy and reproducibility. Analytical validation will be performed in accordance with FDA and Clinical Laboratory Standard Institute (CLSI) recommendations, using a combination of clinically positive patient samples and samples spiked with synthetically generated aggregates to evaluate method accuracy, precision, sensitivity, specificity, reportable range, and other critical assay characteristics. The overall objectives for phase I are to finalize standard operating procedures and protocols and perform analytical validation of qualitative/semi-quantitative αS-PMCA assays for detection of αS oligomers in CSF and plasma for use as a diagnostic biomarker for PD and related synucleinopathies. The overall objectives for phase II are to adapt the assays for quantitation/kinetic characterization of αS oligomers in CSF and plasma, and to explore expanded context of use to include indications as: a susceptibility/risk biomarker to identify patients at risk of developing synucleinopathies prior to onset of clinical symptoms; a diagnostic biomarker to distinguish between different type of synucleinopathies; a monitoring biomarker to assess disease progression or treatment effectiveness; and a prognostic biomarker with ability to characterize disease type and severity.