A Fully Integrated Point-of-Care Test for Ebola

项目摘要 该提案的目的是开发一种新的即时检测（POCT）-D4检测-用于早期现场- 检测埃博拉病毒（EBOV）感染。目前，EBOV是通过使用逆转录病毒的RNA检测来诊断的。 转录酶-聚合酶链反应（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 检测（LFA），缺乏早期检测的敏感性，这对于及时干预可用的 这种抗体混合物可以使低病毒血症患者的存活率达到90%。我们的目标是 临床上迫切需要一种POCT，它（1）能在现场快速可靠地检测EBOV感染，（2） 现场基础设施，（3）在30分钟内产生结果，和（4）匹配或超过RT-PCR的性能。到 为了实现这些目标，我们设计了一种集成的POCT D4测定，其具有四个简单的步骤-分配， 溶解、扩散和检测需要有限处理和技能来执行。这项新的前沿技术 这是一个明确的存在的优势，病毒分泌的糖蛋白sGP存在于血清中， 患者在感染初期。我们已经为sGP生产了定制的单克隆抗体（Abs）， 用于D4试验。我们设计的当前原型D4测定检测至少一种EBOV感染。 在感染猴中，比RT-PCR早一天，成本比RT-PCR或LFA低得多。在这一提议中， 我们计划推进我们的开发，进一步提高D4检测的灵敏度，并减少 我们的策略是增加我们目前的抗体的平衡结合常数， 采用抗体亲和力成熟技术和高通量筛选技术， 抗体对增强的D4检测试剂盒将在蛋白质上打印捕获和检测抗体 以及在玻璃板上的抗细胞聚合物刷，该玻璃板被封装在被动毛细作用微流体芯片中。测定 输出将是D4芯片上微点的荧光。我们开发了一种便携式手持荧光灯 检测器，以捕获和成像斑点，并自动将其转换为分析物的浓度， 定量并上传到安全服务器该设计将进行严格的测试和验证的样品 从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 将有一个现场准备，用户友好，高度敏感的POCT，将允许医护人员检测EBOV 新的设计将推动EBOV目前的边界， 检测和促进更迅速地部署感染控制和病人支持措施， 如果在感染早期实施，可以获得90%的存活率或更高。由于D4 POCT是可复用的，因此它将 这为EBOV以外的更广泛的用途开创了先例，以诊断许多其他传染病。

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.