Development of a Biochemical Diagnosis for Creutzfeldt-Jakob disease

描述（由申请人提供）：人类朊病毒疾病是传染性和不可避免的致命形式的神经退行性疾病，包括散发的克雅氏病（sCJD），最常见的形式，以及变异型CJD (vCJD)，这与食用受牛海绵状脑病感染的牛肉有关。目前还没有对这些疾病进行敏感、客观、无创的生化诊断，更没有在症状前期对潜伏者进行检测的程序。这是公共卫生的一个主要问题，因为已知朊病毒疾病会在人与人之间进行医源性传播，而且由于该疾病的症状前阶段很长（可能长达50年），无症状携带者的数量远远超过临床感染的个体。使情况更加复杂的是，导致这些疾病的传染因子被称为朊病毒，它完全由一种名为PrPSc的自然产生的蛋白质的构象改变形式组成，这种蛋白质具有感染个体并在体内繁殖的独特能力，而不需要遗传物质。PrPSc不仅是神经变性的传染因子和可能的罪魁祸首，而且是该疾病的最佳替代标志物。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以微量存在。该提案的主要目标是发展一种血液-和

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.