Rapid point of-care test for Lyme serodiagnosis based on novel ultra-sensitive de

描述（由申请人提供）：拟议项目旨在开发和实施用于快速医学诊断测试的高灵敏度比色检测技术，并将其应用于基于肽的莱姆病快速测试作为第一个例子。 新技术的核心是一种新的多色显色底物系统，用于广泛使用的酶标记辣根过氧化物酶，利用固定化形式的显色试剂。该技术为酶联免疫测定在开发高灵敏度快速诊断测试中的应用开辟了新的机会。新的检测技术将提供传统技术目前无法达到的灵敏度水平，但保留了传统POC测试的简单性、低成本、可制造性和稳定性。在POC测试中实施新技术将使得有可能开发具有非常短的测定时间的快速测试，但分析灵敏度超过利用常规比色标记（例如胶体金或染色乳胶颗粒）以及微孔板ELISA的测试。此外，灵敏度预计将与目前正在开发的快速检测新的复杂仪器依赖技术的灵敏度相媲美。该技术将适用于当前所有主要形式的快速诊断、侧向流、流过或浸渍条以及珠过滤测定。新的稳定的底物系统具有独特的多路复用能力，将在诊断膜上产生明亮、高对比度的彩色区域，非常适合视觉阅读或密度测定。为了验证新的检测技术在体外诊断试验中的适用性，将开发一种新的莱姆病血清学侧流试验。目前，市场上没有具有临床有用的灵敏度和特异性的莱姆病即时检测。新的快速检测将利用两种肽抗原，VLsE的C6肽和OspC的C10肽，并将提供对疾病早期阶段的诊断灵敏度的显着改善，超过目前的方法，包括微孔板ELISA。快速检测的周转时间为3-5分钟，全血作为样本类型将允许床旁使用。新的快速检测的性能特征将使用来自培养证实或医生记录的早期莱姆病患者和对照人群的良好表征的血清样本进行评估。 公共卫生相关性：该项目旨在开发一种适用于即时诊断测试的新比色检测技术，其灵敏度明显高于现有方法。这项技术将提供一个快速的周转，成本效益的解决方案，为即时检测使用可视标签。作为第一个应用，将基于肽抗原开发莱姆病的快速检测，这将是第一个能够对早期莱姆病进行准确的即时血清学诊断。

DESCRIPTION (provided by applicant): The proposed project is aimed at the development and implementation of a highly sensitive colorimetric detection technology for use in rapid medical diagnostic tests, and its application to a peptide-based Lyme disease rapid test as a first example. At the core of the new technology is a novel multi-color chromogenic substrate system for the widely used enzyme label horseradish peroxidase utilizing immobilized forms of color-generation reagents. This technology opens new opportunities for the application of enzyme -linked immunoassays to development of highly sensitive rapid diagnostic tests. The new detection technology will provide a level of sensitivity which is currently unattainable with conventional technologies, but preserving the simplicity, low cost, manufacturability and stability of conventional POC tests. Implementation of the new technology in POC tests will make it possible to develop rapid tests with very short assay time but with analytical sensitivity exceeding that of tests utilizing conventional colorimetric labels, such as colloidal gold or dyed latex particles, as well as microplate ELISA's. Moreover, the sensitivity is expected to compare favorably with the sensitivities of new sophisticated instrument-dependant technologies currently in development for rapid tests. The technology will be applicable to all major formats of current rapid diagnostic, lateral flow, flow through or dip-strip as well as bead filtration assays. The new stable substrate system with its unique multiplexing capabilities will produce bright, high-contrast, colored zones on diagnostic membranes ideally suited for either visual reading or densitometry. To validate applicability of the new detection technology to in vitro diagnostic tests, a new serological lateral flow test for Lyme disease will be developed. At present, there is no point-of-care test for Lyme disease on the market with clinically useful sensitivity and specificity. The new rapid test will utilize two peptide antigens, the C6 peptide of VLsE and the C10 peptide of OspC, and will provide a significant improvement in the diagnostic sensitivity for early phases of the disease over current methods including microplate ELISA's. Turnaround time for the rapid test will be 3-5 minutes, and whole blood as sample type will allow point-of-care use. Performance characteristics of the new rapid test will be evaluated using well-characterized serum samples from Lyme patients with culture-confirmed or physician documented early stage Lyme disease, and on control populations. PUBLIC HEALTH RELEVANCE: This project is aimed at development of a new colorimetric detection technology suitable for point-of-care diagnostic tests, which offers significantly greater sensitivity than current methods. This technology will provide a rapid turnaround, cost-effective solution for point-of-care testing using multicolor visual labels. As a first application, a rapid test for Lyme disease will be developed based on peptide antigens, which will be the first to enable accurate point-of-care serodiagnosis for early Lyme disease.