Multiplexed immunoassay for building patient-specific molecular profiles of CSF amyloid beta and TAU

摘要 该项目将开发一种多重免疫分析方法，用于测量患者特定的两种病毒的分子图谱。 阿尔茨海默病(AD)的蛋白质标志物：人类脑脊液(CSF)中的淀粉样β蛋白(ABeta)和TAU。 这两种蛋白质由于存在多种差异裂解形式(ABeta)和6 含有50+磷酸化位点(TAU)的异构体。脑脊液提供了丰富的潜在生物标志物来源， 可广泛挖掘以建立AD和AD相关痴呆(ADRD)的诊断和预后特征 或者为药物发现开发个性化的患者档案。目前，这些努力受到 缺乏解决多种ABeta和TAU蛋白形式的技术能力，更具体地说是由于缺乏产品 用于ABeta和TAU的多重捕获和浓缩，用于基于定量质谱学的蛋白质组学。 这项提议寻求建立在早先成功的产品开发项目的基础上，该项目由Adetrix为 一位制药公司的顾客。利用我们的BAMS™平台，我们能够鉴定出至少14个新的低丰度脑脊液 并通过添加两种新类型的探针显著扩展了脑脊液TAU的序列覆盖范围， 它们补充了现有的总(TTAU)和磷酸-TAU(Ptau)的常规探针。建议数 免疫分析，称为ABETA/TauScan™，将满足生物学家、临床医生和制药商目前对 研究神经退行性变的蛋白质标记物的综合分子图谱工具。此外，它还将 为开发其他蛋白质靶标的类似分析开辟了道路，如神经细丝轻多肽(NFL) 和α-突触核蛋白。虽然ABETA/TauScan™主要用于脑脊液，但在第二阶段之后，将对试剂进行评估 用于血清/血浆。 在整个项目中，我们将与学术、临床和行业合作伙伴密切合作，以确保 ABETA/TauScan™的分析性能。一旦建立了化验方法，将通过筛选300例脑脊液进行验证 代表认知正常(CN)、轻度认知障碍(MCI)和AD受试者的样本 建立从CN到MCI到AD进展的分子标记，该分子标记将至少包含一个新的标记 提高了检测的灵敏度和特异性。

ABSTRACT This project will develop a multiplexed immunoassay for measuring patient-specific molecular profiles of the two protein markers of Alzheimer’s disease (AD): amyloid beta (ABeta) and TAU from human cerebrospinal fluid (CSF). Both proteins are structurally complex due to the existence of multiple differentially cleaved forms (ABeta) and 6 isoforms containing 50+ phosphorylation sites (TAU). CSF provides a rich source of potential biomarkers, which can be extensively mined to build diagnostic and prognostic signatures of AD and AD-related dementias (ADRD) or to develop personalized patient profiles for drug discovery. At present, those efforts are constrained by the lack of technical capability to resolve multiple ABeta and TAU proteoforms, more specifically by the lack of products for multiplexed capture and enrichment of ABeta and TAU for quantitative mass spectrometry-based proteomics. This proposal seeks to build upon an earlier successful product development project performed by Adeptrix for a pharma customer. Using our BAMS™ platform, we were able to identify at least 14 novel low abundance CSF ABeta peptides and dramatically expand the sequence coverage of CSF TAU by adding two new types of probes, which complement the existing conventional probes for total (tTAU) and phospho-TAU (pTAU). The proposed immunoassay, termed ABeta/TauScan™ will address the current need of biologists, clinicians, and pharma for comprehensive molecular profiling tools for studying protein markers of neurodegeneration. Furthermore, it will open a path to developing similar assays for other protein targets, such as neurofilament light polypeptide (NFL) and alpha-synuclein. While ABeta/TauScan™ is intended primarily for CSF, post Phase II the reagents will be evaluated for use in serum/plasma. Throughout this project we will work closely with academic, clinical and industry partners to ensure the robust analytical performance of ABeta/TauScan™. Once the assay is created, it will be validated by screening 300 CSF samples representing cognitively normal (CN) subjects, mild cognitive impairment (MCI) and AD subjects and establishing a molecular signature of CN to MCI to AD progression that will contain at least one novel marker for improved assay sensitivity and specificity.