Development of a Biochemical Diagnosis for Creutzfeldt-Jakob disease

克雅氏病生化诊断的发展

• 批准号: 8727118
• 负责人: RUSSELL M LEBOVITZ
• 金额: $ 50.14万
• 依托单位: AMPRION, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727118
• 关键词: Affect; Age; Animals; Antibodies; Autopsy; Biochemical; Biological; Biological Assay; Biological Availability; Blinded; Blood; Blood Tests; Blood Transfusion; Blood specimen; Bovine Spongiform Encephalopathy; Brain; Businesses; Cattle; Cerebrospinal Fluid; Clinical; Code; Communicable Diseases; Consultations; Consumption; Creutzfeldt-Jakob Syndrome; Decontamination; Detection; Development; Diagnosis; Diagnostic tests; Disease; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Genetic Materials; Goals; Guidelines; Hamsters; Health; Human; Incubated; Individual; Infectious Agent; International; Knockout Mice; Laboratories; Lead; Liquid substance; Meat; Medical; Methods; Monoclonal Antibodies; Mus; Names; Nerve Degeneration; Neurodegenerative Disorders; Non-Invasive Cancer Detection; Patients; Peripheral; Peritoneal; Phase; PrP; PrPSc Proteins; Prion Diseases; Prions; Procedures; Production; Proteins; Public Health; Reproducibility; Resistance; Rodent; Sampling; Scrapie; Secure; Sensitivity and Specificity; Signal Transduction; Small Business Technology Transfer Research; Specificity; Spleen; Staging; Sterilization; Surrogate Markers; Symptoms; Techniques; Technology; Testing; Time; Tissues; Transplantation; Urine; Validation; Variant; Work; authority; base; clinical Diagnosis; commercialization; design; disease diagnosis; infected vector rodent; programs; protein misfolding cyclic amplification; research study; single molecule

DESCRIPTION (provided by applicant): Human prion diseases are infectious and invariably fatal forms of neurodegenerative diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, and variant CJD (vCJD) which is associated to consumption of cattle meat infected by bovine spongiform encephalopathy. Currently there is not sensitive, objective and non-invasive biochemical diagnosis for these diseases, and even less a procedure to detect people incubating the disease in the pre-symptomatic period. This is a major problem for public health, because prion diseases are known to transmit iatrogenically between human-to-human and because due to the long pre-symptomatic stage of the disease-which may span five decades-the asymptomatic carriers far outnumber the clinically affected individuals. To make the situation even more complicated, the infectious agent responsible for these diseases, termed prion, is composed exclusively of a conformationally altered form of a naturally occurring protein, named PrPSc, which has the unique ability to infect individuals and propagate in the body without the need for genetic material. PrPSc is not only the infectious agent and the likely culprit of neurodegeneration, but also the best surrogate marker for the disease. The challenge is that its quantity is high only in the brain at late stages of the disease However, compelling evidences indicate that PrPSc is present in minute amounts in various peripheral tissues and biological fluids. The main goal of this proposal is to develop a blood- and cerebrospinal fluid (CSF)-based detection assay for PrPSc associated with sCJD and vCJD. Our strategy utilizes two pioneering proprietary technologies developed in our lab: First the protein misfolding cyclic amplification (PMCA) technique which enables to amplify the amount of PrPSc present in the sample to detect as little as a single molecule of PrPSc. PMCA, has a similar power of amplification as PCR and allowed us to detect, for the first time, prions in blood and urine, even at the pre-symptomatic stages of the disease. Second, PrPSc-specific monoclonal antibodies (called PrioC) raised against prion-infected brain homogenates in PrP knock out mice. In this project we will optimize a technology combining PMCA amplification of PrPSc with detection by sandwich ELISA using the PrioC conformational antibodies. The assay will be optimized using animal and human samples for high throughput detection of prions in biological fluids, and will be validated for sensitivity and specificity according to the requiremets of the regulatory authorities with the aim to obtain approval for commercialization. The results generated in this project may lead to the first biochemical test validated for the diagnosis of CJD. With this validated technology, Amprion will establish an International Reference Laboratory for the Detection and Eradication of human prion diseases.

描述（由申请人提供）：人类朊病毒病是一种传染性且总是致命的神经退行性疾病，包括最常见的散发性克雅氏病 (sCJD)，以及与食用受牛海绵状脑病感染的牛肉有关的变异型克雅氏病 (vCJD)。目前，这些疾病还没有灵敏、客观、无创的生化诊断方法，更没有在症状发生前检测潜伏期的方法。这是公共卫生的一个主要问题，因为已知朊病毒疾病可以在人与人之间医源性传播，而且由于该疾病的症状前阶段很长（可能跨越五十年），无症状携带者的数量远远超过临床受影响的个体。使情况变得更加复杂的是，导致这些疾病的感染因子（称为朊病毒）完全由一种名为 PrPSc 的天然蛋白质的构象改变形式组成，它具有感染个体并在体内传播的独特能力，而不需要遗传物质。 PrPSc 不仅是传染源和神经退行性变的可能罪魁祸首，而且也是该疾病的最佳替代标志物。挑战在于，其数量仅在疾病晚期的大脑中较高。然而，令人信服的证据表明，PrPSc 在各种外周组织和生物体液中存在微量。该提案的主要目标是开发一种血液和 基于脑脊液 (CSF) 的与 sCJD 和 vCJD 相关的 PrPSc 检测分析。我们的策略利用了我们实验室开发的两项开创性专有技术：首先是蛋白质错误折叠循环扩增 (PMCA) 技术，该技术能够放大样品中存在的 PrPSc 量，以检测小至单个 PrPSc 分子。 PMCA 具有与 PCR 相似的扩增能力，使我们能够首次检测到血液中的朊病毒 和尿液，即使是在疾病症状出现前的阶段。其次，在 PrP 敲除小鼠中针对感染朊病毒的脑匀浆产生 PrPSc 特异性单克隆抗体（称为 PrioC）。在该项目中，我们将优化将 PrPSc 的 PMCA 扩增与使用 PrioC 构象抗体通过夹心 ELISA 检测相结合的技术。该检测方法将使用动物和人类样本进行优化，以高通量检测生物体液中的朊病毒，并将根据监管机构的要求验证灵敏度和特异性，以期获得商业化批准。该项目产生的结果可能会导致第一个经过验证的生化测试可用于诊断克雅氏病。凭借这项经过验证的技术，Amprion 将建立一个检测和根除人类朊病毒疾病的国际参考实验室。