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

项目摘要 错误折叠的蛋白质聚集体是有毒的超分子蛋白质聚集体， 在宿主体内复制，对周围组织造成损伤。这些蛋白质聚集体中的几种是 与神经退行性疾病密切相关，例如朊病毒疾病中的朊病毒蛋白， 阿尔茨海默病（AD）中的淀粉样蛋白β和tau，肌萎缩侧索硬化症（ALS）中的TDP-34， 额颞叶痴呆（FTD）和α-突触核蛋白（αS）在帕金森病（PD）、痴呆伴 路易体（DLB）和多发性系统性萎缩（MSA）。这背后的分子机制 这些聚集体的体内形成及其对神经组织的影响仍然是难以捉摸的，但它们的 令人信服的意义，因为疾病过程的早期驱动因素将其确定为潜在有用的 疾病生物标志物和治疗靶点。该提案的主要目标是执行稳健的 用于检测αS的蛋白质错误折叠循环扩增（PMCA）试验的分析验证 突触核蛋白病患者脑脊液（CSF）和血浆中的聚集体。PMCA 该平台利用错误折叠的蛋白质聚集体的内在自我复制性质来循环扩增 生物样品中微量的αS聚集体。在96孔板中进行测定， 并且使用荧光淀粉样蛋白特异性染料ThioflavinT随时间监测扩增。 方法开发的重点是耐用性，以支持未来在受控实验室中的使用， 高样品通量和对测定准确性和再现性的严格要求。分析 将根据FDA和临床实验室标准协会（CLSI）进行确认 建议，使用临床阳性患者样本和加标 合成生成的聚合物，以评价方法的准确度、精密度、灵敏度、专属性， 可报告范围和其他关键检测特性。第一阶段的总体目标是 最终确定标准操作程序和方案，并进行分析验证 用于检测CSF和血浆中αS寡聚体的定性/半定量αS-PMCA测定， 用作PD和相关突触核蛋白病的诊断生物标志物。阶段的总体目标 II旨在调整用于定量/动力学表征CSF和血浆中αS寡聚体的试验， 并探索扩大的使用范围，以包括以下适应症： 在临床症状发作之前识别处于发展突触核蛋白病的风险中的患者; a 用于区分不同类型的突触核蛋白病的诊断生物标志物; 用于评估疾病进展或治疗有效性的生物标志物;以及具有以下特征的预后生物标志物： 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.