A Fully Integrated Point-of-Care Test for Ebola

项目总结 这项建议的目标是开发一种新的护理点检测(POCT)-D4检测-用于早期现场检查。 检测埃博拉病毒(EBOV)感染。目前，EBOV是通过使用反向RNA检测来诊断的 转录酶-聚合酶链式反应(RT-PCR)。RT-PCR需要一个临时的BSL-4级实验室 现场，昂贵的设备和训练有素的人员。其他POCT，包括用户友好的横向流动 检测(LFA)缺乏早期检测的敏感性，而早期检测对于及时干预可用 抗体鸡尾酒，为低病毒血症患者提供90%的存活率。我们的目标是由一个 临床迫切需要(1)现场快速可靠地检测EBOV感染的POCT，(2)几乎不需要 现场基础设施，(3)在30分钟内产生结果，以及(4)达到或超过RT-PCR的性能。至 为了实现这些目标，我们设计了一种集成的POCT D4检测方法，它有四个简单的步骤-分配， 溶解、扩散和检测需要有限处理和技能才能执行的任务。这项新的前沿技术需要 存在一种明确的、病毒分泌的糖蛋白Sgp的优势 感染患者很早就被感染了。我们已经为SGP产生了定制的单抗(Abs)来 在D4试验中使用。我们设计的当前原型D4检测方法至少检测到一种EBOV感染 在感染的猴子中比RT-PCR早一天，而且成本比RT-PCR或LFA低得多。在这份提案中， 我们计划进一步推进我们的开发，提高D4检测的灵敏度，并减少 检测时间为60min~30min。我们的策略是增加我们目前的抗体的平衡结合常数 用抗体亲和成熟技术和高通量筛选从~10-9M到~10-11M的一对 抗体对。增强的D4检测试剂盒将在蛋白质上喷墨打印捕获和检测抗体 以及装在无源毛细微流控芯片中的玻璃板上的抗细胞聚合物刷子。化验结果 输出将是D4芯片上微点的荧光。我们已经开发了一种便携式手持荧光灯 探测器捕获斑点并将其自动转换为分析物的浓度，以便 量化并上传到安全服务器。该设计将经过严格的测试，并通过样例进行验证 来自受感染的人类细胞和实验室挑战的非人类灵长类动物。在这个项目完成后，我们 将有一个现场就绪、用户友好和高度敏感的POCT，使医护人员能够检测到EBOV 在30分钟内在血清、血液或其他体液中。新的设计将突破EBOV目前的界限 检测并促进更便捷地部署感染控制和患者支持措施，以 如果在感染早期实施，存活率可达到90%或更高。因为D4 POCT是可多路传输的，所以它将 为EBOV以外的更广泛的用途诊断许多其他传染病开创了先例。

PROJECT SUMMARY The objective of this proposal is to develop a new point-of-care test (POCT)—the D4 assay—for early field- detection of Ebola virus (EBOV) infection. Currently, EBOV is diagnosed by RNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR requires a makeshift BSL-4 grade laboratory in the field, expensive equipment, and highly trained personnel. Other POCTs, including the user-friendly lateral-flow assay (LFA), lack the sensitivity for early detection that is critical for timely intervention with the available antibody cocktail that yields 90% survival rates for patients with low viremia. Our objective is motivated by an urgent clinical need for a POCT that (1) detects EBOV infection in the field quickly and reliably, (2) requires little on-field infrastructure, (3) yields results in 30 min, and (4) matches or exceeds the performance of RT-PCR. To achieve these goals, we have designed an integrated POCT the D4 assay that has four simple steps—dispense, dissolve, diffuse, and detect that require limited handling and skill to perform. This new-frontier technology takes advantage of the presence of an unmistakable, viral secreted glycoprotein sGP that is present in the serum of infected patients very early in infection. We have generated customized monoclonal antibodies (Abs) for sGP to use in the D4 assay. The current prototype D4 assay that we have designed detects EBOV infection at least one day earlier than RT-PCR in infected monkeys and at a far lower cost than RT-PCR or LFA. In this proposal, we plan to advance our development and improve the sensitivity of the D4 assay further as well as reduce the assay time from 60 min to 30 min. Our strategy is to increase the equilibrium binding constant of our current Ab pair from ~10-9 M to ~10-11 M with antibody affinity maturation techniques and high-throughput screening of antibody pairs. The enhanced D4 assay kit will have inkjet-printed capture and detection antibodies on a protein and cell-resistant polymer brush on a glass plate encased in a passive capillarity microfluidics chip. The assay output will be fluorescence of microspots on the D4 chip. We have developed a portable handheld fluorescence detector to capture and image the spots and automatically convert them into the concentration of analytes for quantitation and uploaded to a secure server. The design will be rigorously tested and validated with samples from infected human cells and laboratory-challenged non-human primates. At the completion of this project, we will have a field-ready, user-friendly, and highly sensitive POCT that will allow healthcare workers to detect EBOV in serum, blood, or other bodily fluids in 30 min. The new design will push the current boundaries of EBOV detection and facilitate more expedient deployment of infection control and patient support measures that can yield 90% survival rates or better if implemented early in infection. Because the D4 POCT is multiplexable, it will set a precedent for broader utility beyond EBOV to diagnose many other infectious diseases.