Ante-mortem Diagnostics for Prion Infection

描述（由申请人提供）：Vegrandis提出开发一种快速、灵敏、高通量、廉价且易于使用的系统，用于朊病毒感染的生前诊断。所提出的系统提供了一个全自动化的设备与一次性微电化学盒的诊断生前水平低至1 × 10-15摩尔（可能为1 × 10- 21摩尔）浓度的PrPSc在第30分钟的快速测定中，高通量允许每个全自动仪器> 100个样品/h，高灵敏度，可靠性> 95%（消除假阳性和假阴性），处理患者生物液体的潜在危险最小。与现有的朊病毒感染诊断方法不同，现有的朊病毒感染诊断方法仅在患者已经显示出神经功能障碍的急性体征时才可用，并且在死后组织匀浆诊断排除或确认诊断之前是临时的，所提出的系统预期诊断无症状阶段的朊病毒感染，其可用于至少三个重要目的：1）消除被污染的动物和动物产品进入市场; 2）用于开发和监测正在开发的药物、疫苗或其他治疗分子的功效;和3）监测药物治疗期间PrPSc水平的变化。所提出的系统将通过在一次性盒（DC）格式的50微米直径威尔斯孔中使用自含式微电化学进行无症状和生前诊断，从而消除使用死后组织匀浆进行诊断。将样品注入DC上的自密封入口端口，将DC插入全自动仪器，将试剂放置在试剂模块中，并选择适当的程序。仪器读数将在<30分钟结束时显示PrPSc的浓度。在第一阶段，Vegrandis将通过使用羊瘙痒病作为模型分析物进行实验来证明技术概念验证。阶段I预期：1）检测低至飞摩尔水平的PrPSc，2）从捕获到信号产生<45分钟的测定，3）来自生物样品的碎片的非干扰，消除了繁琐的样品制备步骤，以及4）由于使用小试剂体积（对于整个微阵列d10 <L或每个微孔约0.5 <L）而导致的低测定成本。第一阶段里程碑的实现将把研究推进到第二阶段，在第二阶段，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 x 10-21摩尔PrPSc检测，以及c）开发一种100通道皮安电化学分析仪，用于每小时100个样品分析的总测定时间。相同的仪器和一次性盒将在固定的或移动的诊所或医院中用作朊病毒疾病的早期检测、生前和死后检测的诊断工具。随着关于朊病毒及其抗体的更多细节的获得，它们将被纳入一次性检测盒中以供进一步研究。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。在2004年发现第一例国内疯牛病病例后，仅在那一年，美国全国牛肉行业的损失就达到了32亿至47亿美元。全世界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目前没有疫苗、解毒剂或药物可治愈的致命疾病的传播。因此，避免污染至关重要。开发一种全自动，便携式仪器，使用紧凑，一次性盒，不需要分析前的样本制备的死前朊病毒感染的建议。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的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.