A Fully Integrated Point-of-Care Test for Ebola

完全集成的埃博拉即时护理测试

• 批准号: 10686305
• 负责人: Ashutosh Chilkoti
• 金额: $ 59.02万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-23 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686305
• 关键词: 3-Dimensional; Address; Adsorption; Affinity; Antibodies; Antibody Affinity; Binding; Biological Assay; Blood; Blood capillaries; Blood specimen; Body Fluids; Capillarity; Cartoons; Cell model; Cells; Clinical; Communicable Diseases; Detection; Development; Devices; Diagnosis; Diffuse; Diffusion; Disease Outbreaks; Dissociation; Drops; Early Diagnosis; Ebola; Ebola virus; Enzyme-Linked Immunosorbent Assay; Epidemic; Epitopes; Equilibrium; Equipment; Exposure to; Film; Fluorescence; Glass; Glycoproteins; Goals; Health Personnel; Human; Human Resources; Image; Incubated; Infection; Infection Control; Infrastructure; Intervention; Label; Laboratories; Macaca; Macaca fascicularis; Macaca mulatta; Measures; Methods; Microfluidic Microchips; Microfluidics; Modeling; Monkeys; Monoclonal Antibodies; Mutate; Output; Patients; Performance; Pharmaceutical Preparations; Polymers; Power Sources; Printing; Proteins; RNA; Reader; Reagent; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Running; Sampling; Secure; Serum; Speed; Spottings; Sudan; Surface; Survival Rate; Techniques; Technology; Testing; Time; Time Study; Training; Translating; Viral; Viral Proteins; Viremia; Virus Diseases; Zaire Ebola virus; antibody detection; clinical diagnostics; cost; design; detection limit; detector; fluorescence imaging; frontier; high throughput screening; imaging detector; imaging platform; improved; lateral flow assay; nonhuman primate; point of care; point of care testing; portability; protein biomarkers; prototype; skills; synergism; user-friendly; viral RNA; viral detection; virtual; wireless transmission

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.

项目摘要 该提案的目的是为早期领域开发新的护理测试（POCT） - D4分析 - 检测埃博拉病毒（EBOV）感染。目前，通过使用反向检测来诊断EBOV 转录酶 - 聚合酶链反应（RT-PCR）。 RT-PCR需要临时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程 测定（LFA），缺乏对早期检测的敏感性，这对于及时干预至关重要 低病毒血症患者的抗体鸡尾酒可产生90％的存活率。我们的目标是由 紧急临床需求（（1）迅速可靠地检测到现场的EBOV感染，（2）几乎不需要 现场基础架构（3）在30分钟内产生结果，（4）匹配或超过RT-PCR的性能。到 实现这些目标，我们设计了一个集成的POCT D4测定法，该测定法具有四个简单的步骤：dispense， 溶解，扩散和检测需要有限的操作和技巧才能执行。这项新技术需要 存在于血清中存在的明确，病毒分泌的糖蛋白SGP的优势 感染很早就感染患者。我们已经生成了SGP的自定义单克隆抗体（ABS） 在D4测定中使用。当前我们设计的当前原型D4分析检测到EBOV感染至少一种 在感染猴子中，比RT-PCR早于RT-PCR，其成本比RT-PCR或LFA低得多。在此提案中， 我们计划提高我们的发展并进一步提高D4分析的敏感性，并减少 分析时间从60分钟到30分钟。我们的策略是增加当前AB的等效结合常数 通过抗体亲和力成熟技术从〜10-9 m到〜10-11 m的成对 抗体对。增强的D4测定套件将在蛋白质上具有喷墨打印的捕获和检测抗体 在被动毛细血管微流体芯片中包裹的玻璃板上的抗细胞聚合物刷。测定法 输出将是D4芯片上微量司的荧光。我们已经开发了一种便携式手持式荧光 检测器捕获和成像斑点，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。该设计将经过严格测试并用样品验证 来自被感染的人类细胞和实验室挑战的非人类素数。该项目完成时，我们 将有一个现场准备，用户友好且高度敏感的POCT，这将使医疗人员能够检测到Ebov 在30分钟内血清，血液或其他体液。新设计将推动Ebov的当前边界 检测并促进更方便地部署感染控制和患者支持措施 如果在感染早期实施，则产生90％的存活率或更高的生存率。因为D4 POCT是多重的，它将 为EBOV以外的更广泛的实用程序设定了诊断许多其他传染病的先例。