A Fully Integrated Point-of-Care Test for Ebola

完全集成的埃博拉即时护理测试

基本信息

批准号：
10269019
负责人：
Ashutosh Chilkoti
金额：
$ 71.74万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10269019
关键词：
3-Dimensional Address Adsorption Affinity Antibodies Antibody Affinity Binding Biological Assay Blood Blood capillaries Blood specimen Bundibugyo virus Capillarity Cartoons Cell model Cells Clinical Communicable Diseases Custom 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 Gold Hand Health Personnel Human Human Resources Image Incubated Infection Infection Control Infrastructure Intervention Label Laboratories Liquid substance 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 Spottings Sudan Sudan Ebola virus Surface Survival Rate Techniques Technology Testing Time Time Study Training Translating Viral Viral Proteins Viremia Virus Diseases Wireless Technology Zaire Ebola virus antibody detection base clinical diagnostics cost design detection assay 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 user-friendly viral RNA viral detection virtual

项目摘要

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.

项目概要该提案的目标是开发一种新的即时检测 (POCT)——D4 检测——用于早期现场检测。检测埃博拉病毒（EBOV）感染目前，埃博拉病毒是通过使用反向RNA检测来诊断的。转录酶聚合酶链式反应 (RT-PCR) 需要在实验室内建立一个临时 BSL-4 级实验室。其他 POCT，包括用户友好的侧流层析。分析（LFA），缺乏早期检测的敏感性，这对于及时干预可用的方法至关重要我们的目标是让低病毒血症患者的存活率达到 90%。临床迫切需要一种 POCT，其 (1) 在现场快速可靠地检测 EBOV 感染，(2) 几乎不需要现场基础设施，(3) 在 30 分钟内产生结果，(4) 匹配或超过 RT-PCR 的性能。为了实现这些目标，我们设计了一种集成的 POCT D4 检测，它有四个简单的步骤：分配、溶解、扩散和检测需要有限的操作和技能来执行。其优点是存在于血清中的明确无误的病毒分泌糖蛋白 sGP 我们已经为 sGP 生成了定制的单克隆抗体 (Ab)。我们设计的当前 D4 检测原型可检测至少一种 EBOV 感染。在受感染的猴子中，比 RT-PCR 早一天，并且成本比 RT-PCR 或 LFA 低得多。我们计划推进我们的开发，进一步提高 D4 测定的灵敏度，并减少检测时间从 60 分钟延长至 30 分钟我们的策略是增加当前抗体的平衡结合常数。通过抗体亲和力成熟技术和高通量筛选，从 ~10-9 M 配对到 ~10-11 M 增强型 D4 检测试剂盒将在蛋白质上喷墨打印捕获和检测抗体。以及封装在被动毛细管微流体芯片中的玻璃板上的细胞抗性聚合物刷。输出将是 D4 芯片上微点的荧光。我们开发了便携式手持式荧光。检测器捕获斑点并对其成像，并自动将其转换为分析物的浓度该设计将通过样品进行严格测试和验证。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。将拥有现场就绪、用户友好且高度敏感的 POCT，使医护人员能够检测 EBOV 新设计将在 30 分钟内突破血清、血液或其他体液中的 EBOV。检测并促进更便捷地部署感染控制和患者支持措施，这些措施可以如果在感染早期实施，则可实现 90% 或更高的存活率，因为 D4 POCT 是可多重使用的，因此它会。开创了除埃博拉病毒之外更广泛用途以诊断许多其他传染病的先例。