Ante-mortem Diagnostics for Prion Infection

描述（由申请人提供）：Vegrandis拟开发一种快速、敏感、高通量、廉价且易于使用的系统，用于朊病毒感染的死前诊断。该系统提供了一种全自动设备，配有一次性微电化学盒，可在30分钟内快速诊断死前低至1 × 10-15摩尔（可能是1 × 10-21摩尔）浓度的PrPSc，高通量允许每台全自动仪器100个样品/小时，高灵敏度，可靠性为> 95%（消除假阳性和假阴性），并且处理患者生物液体的潜在危害最小。现有的朊病毒感染诊断方法只有在患者已经表现出急性神经功能障碍的迹象时才能使用，并且在死后组织匀浆诊断排除或确认诊断之前是暂时的，与这种方法不同，拟议的系统有望诊断无症状阶段的朊病毒感染，可用于至少三个重要目的：1)消除受污染的动物和动物产品进入市场；（二）用于研制和监测正在研制的药物、疫苗或者其他治疗性分子的疗效；3)监测用药过程中PrPSc水平的变化。该系统将通过在50微米直径的一次性药筒（DC）中使用独立的微电化学来进行无症状和死前诊断，从而消除使用死后组织匀浆进行诊断的问题。将样品注入DC上的自密封进样口，将DC插入全自动仪器，将试剂放置在试剂模块中，选择合适的程序。仪器读数将在< 30min结束时显示PrPSc的浓度。在第一阶段，Vegrandis将通过使用痒病作为模型分析物进行实验来演示技术的概念验证。第一阶段预计将：1)检测低至飞摩尔水平的PrPSc， 2)从捕获到信号产生的分析时间<45分钟，3)生物样品碎片的无干扰，消除繁琐的样品制备步骤，4)由于使用小试剂量（整个微阵列的d 10 <L或每微孔约0.5 <L），因此分析成本低。第一阶段里程碑的实现将推动研究进入第二阶段，我们设想a)开发一种小型化流动注射仪器，用于无症状朊病毒感染的高通量全自动检测，b)实现1 × 10-21摩尔PrPSc检测，以及c)开发100通道皮安电化学分析仪，每小时总分析时间为100个样品。同样的仪器和一次性药筒将用于固定或流动诊所或医院，作为早期发现、死前和死后检测朊病毒疾病的诊断工具。随着有关朊病毒及其抗体的更多细节的获得，它们将被纳入一次性药筒中以供进一步研究。公共卫生相关性：在美国，每年有数百万头牛被屠宰作为食物。2004年美国国内发现首例疯牛病病例后，仅当年一年，美国牛肉产业的损失就高达32亿至47亿美元。世界各地都有人因食用受感染的牛肉而死于朊病毒感染。死前检测朊病毒感染将防止这种致命疾病的传播，目前尚无疫苗、解毒剂或药物可治愈。因此，避免污染是至关重要的。建议开发一种全自动便携式仪器，该仪器使用紧凑的一次性药盒，不需要对死前朊病毒感染进行预分析样品制备。初步工作表明，所提出的方法将能够满足诊断这种致命疾病无症状阶段所需的高灵敏度。

DESCRIPTION (provided by applicant): Vegrandis proposes to develop a rapid, sensitive, high throughput, inexpensive, and easy to use system for the ante-mortem diagnosis of prion infection. The proposed system offers a fully automated equipment with a disposable microelectrochemical cartridge for diagnosis of ante- mortem levels as low as 1 x 10-15 molar (possibly 1 x 10-21molar) concentrations of PrPSc in a rapid assay at d 30 min, high throughput allowing > 100 samples/h per fully automated instrument, high sensitivity with > 95% reliability (eliminating false positives and false negatives), and with minimal potential hazard of handling patient biological fluid. Unlike existing diagnostic methods of prion infection which are usable only when patients already show acute signs of neurologic dysfunctions and are provisional until post-mortem tissue homogenate diagnosis rules out or confirms the diagnosis, the proposed system is anticipated to diagnose asymptomatic stage prion infection that can be used for at least three important purposes: 1) eliminate contaminated animals and animal products from entering the market; 2) use in developing and monitoring the efficacy of drugs, vaccine, or other therapeutic molecules that are being developed; and 3) monitor changes in the PrPSc levels during medication. The proposed system will eliminate the use of post mortem tissue homogenates for diagnosis by allowing asymptomatic and ante-mortem diagnosis through the use of self-contained microelectro- chemistry in 50-micron diameter wells in a disposable cartridge (DC) format. The sample will be injected into the self-sealing inlet port on the DC, the DC will be inserted into the fully automated instrument, the reagents will be placed in the reagent module, and the proper program will be chosen. The instrument read out will display the concentration of PrPSc at the end of <30 min. During Phase I, Vegrandis will demonstrate the technology proof-of-concept by performing experiments using scrapie as the model analyte. Phase I is anticipated to:1) detect as low as femtomolar levels of PrPSc, 2) <45 min assay from capture to signal generation, 3) non- interference of debris from biological samples eliminating tedious sample preparation steps, and 4) low assay cost resulting from the use of small reagent volumes (d 10 <L for the entire microarray or ~0.5 <L per microwell). Achievement of the milestones in Phase I will advance the research to Phase II where we envision to a) develop a miniaturized flow injection instrument a high throughput fully automated detection of asymptomatic prion infection, b) achieve 1 x 10-21 molar PrPSc detection, and c) develop a 100-channel pico-ampere electrochemical analyzer for 100 sample analysis per hour total assay time. The same instruments and disposable cartridge will have use in stationary or mobile clinics or hospitals as a diagnostic tool for the early detection, ante- and post-mortem detection of prion disease. As additional details about prions and their antibodies become available, they will be incorporated into the disposable cartridge for further study. PUBLIC HEALTH RELEVANCE: Millions of cows are slaughtered annually for food in the United States. After the discovery of the first domestic case of mad cow disease in 2004, national US beef industry losses were recorded at $3.2 to $4.7 billion for that year alone. People have died worldwide from prion infection resulting from eating infected beef. Ante-mortem detection of prion infection will prevent the spread of this fatal disease that currently has no vaccine, antidote, or medicine to cure. Therefore, avoiding contamination is essential. Development of a fully automated, portable instrument that uses compact, disposable cartridges and requires no pre-analysis sample preparation for ante-mortem prion infection is proposed. Preliminary work indicates that the proposed approach will be able to meet the high sensitivity required to diagnose the asymptomatic stage of this fatal disease.