Highly-sensitive zika and dengue antigen/antibody combo test for the point of care

用于护理点的高灵敏度寨卡和登革热抗原/抗体组合测试

• 批准号: 9347812
• 负责人: Marta Fernandez-Suarez
• 金额: $ 22.17万
• 依托单位: DAKTARI DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9347812
• 关键词: Affinity; Anodes; Antibodies; Antigens; Arboviruses; Bedside Testings; Benchmarking; Biological Assay; Biological Markers; Blood; Chemistry; Clinical; Dengue; Dengue Infection; Dengue Virus; Detection; Diagnosis; Diagnostic tests; Disease Management; Disease Outbreaks; Enzyme-Linked Immunosorbent Assay; Flavivirus; Formulation; Future; Goals; Health; Hepatitis C; Immunization; Immunoassay; Immunoglobulin M; Infection; Label; Laboratories; Lateral; Liquid substance; Magnetism; Microfluidics; Modification; Motor; Mus; Noise; Nucleic Acid Amplification Tests; Performance; Phase; Plasma; Preparation; Prevention; Proteins; Protocols documentation; Reagent; Research Infrastructure; Resources; Sampling; Sensitivity and Specificity; Serotyping; Signal Transduction; Silver; Source; Specificity; Symptoms; System; Technology; Temperature; Testing; Training; Training and Infrastructure; Update; Viral Antigens; Viral Proteins; Virus; Zika Virus; base; cross reactivity; design; high risk; instrument; manufacturing process; nanoparticle; particle; point of care; point-of-care diagnostics; prevent; programs; rapid diagnosis; response; screening; viral detection

Project Summary A simple point-of-care (POC) diagnostic test, with laboratory-level performance and the ability to detect the virus across the active phase of infection, is vital to efficient prevention and response efforts during the current Zika virus outbreak, and future management of the disease as it spreads. The ability to differentiate between Zika and Dengue viruses (ZIKV, DENV) is also of utmost importance, particularly in low-resource settings where Dengue is endemic. While lateral flow immunoassays (LFIs) to detect IgM antibodies are deployable, they cannot detect early infection (0-7 days post-onset of symptoms, DPO). On the other hand, the lack of infrastructure and trained lab staff prevents wide use of highly sensitive nucleic acid tests (NATs). There is a need for a simple POC test that is highly sensitive and can detect infection during the entire period of active infection (0-90 DPO). Daktari proposes to develop a simple POC ZIKV/DENV Ag/Ab combo test sensitive and specific enough to detect and differentiate Zika and Dengue infections. By combining detection of viral antigen with host IgM antibodies, we extend the use window of the test to the entire active infection period. Non-structural 1 (NS1) antigen has been validated as a biomarker for arboviruses and NS1 antigen tests exist for Dengue; their sensitivity, however, is poor when implemented at the POC. No Zika NS1 Ag tests have been developed to date. In this Fast-Track program, we will adapt Daktari's POC Virus and Protein (ViPr) platform to detect ZIKV and DENV NS1 and IgM in blood. ViPr consists of (1) Daktari's core technology for automated microfluidic sample preparation (load 25-100 μL of fingerprick blood into the cartridge - insert cartridge into instrument); (2) a high-sensitivity immunoassay detection technology based on silver nanoparticle labels and anodic stripping voltammetry, proven to enable limits of detection (LOD) ≤1 pg/mL protein in plasma (3 logs better than LFAs); and (3) a simple, robust instrument with built-in connectivity and a self-contained disposable cartridge. We will first demonstrate the feasibility of adapting Daktari's viral protein detection assay to the detection of Zika and Dengue antigens. Phase I will focus on developing and screening high-affinity and specific antibodies against the non-structural 1 (NS1) antigens of ZIKV and DENV. We will then conjugate selected antibodies to the magnetic and silver particles, and we will optimize the assay protocol. Finally, we will evaluate assay performance in plasma samples. In Phase II, we will develop the Daktari ViPr IgM assay, and then adapt the ViPr cartridge to implement the ZIKV/DENV NS1 and IgM assays. We will also upgrade the Daktari instrument to incorporate enough actuators to perform the four parallel assays. Finally, performance of the integrated test will be evaluated in clinical samples. With the simple-to-use and sensitive Daktari ZIKV/DENV Ab/Ag combo system, it will be possible for health workers with minimal training to diagnose active Zika infection quickly and accurately, anywhere in the world.

项目摘要 一个简单的护理点（POC）诊断测试，具有实验室级的性能和检测能力 整个感染活性阶段的病毒对于当前的有效预防和反应工作至关重要 寨卡病毒爆发，并在疾病传播时对这种疾病的未来管理。区分能力 Zika和登革热病毒（Zikv，DENV）也很重要，尤其是在低资源环境中 登革热是内在的。 虽然可以部署IgM抗体的侧向流免疫测定（LFI），但它们无法提早检测到 感染（症状发出后0-7天，DPO）。另一方面，缺乏基础设施和训练有素的实验室 工作人员可以防止广泛使用高度敏感的核酸测试（NAT）。需要一个简单的POC测试 高度敏感，可以在整个活性感染期间（0-90 DPO）检测感染。 达克塔里（Daktari 检测并区分寨卡病毒和登革热感染。通过将病毒抗原与宿主IgM的检测结合在一起 抗体，我们将测试的使用窗口扩展到整个活动感染周期。非结构1（NS1） 抗原已被证实为arboviruse的生物标志物，登革热存在NS1抗原测试。他们的 但是，在POC实施时，敏感性很差。迄今为止尚未开发Zika NS1 AG测试。 在这个快速轨道程序中，我们将适应Daktari的POC病毒和蛋白质（VIPR）平台来检测ZIKV和 血液中的DENV NS1和IgM。 VIPR由（1）Daktari的自动微流体样品的核心技术组成 制备（将25-100μl的指纹血液加入弹药筒中 - 将墨盒插入仪器中）； （2）a 基于银纳米颗粒标签和阳极剥离的高敏性免疫测定检测技术 伏安法，证明可以实现等离子体中检测的限制（LOD）≤1pg/ml蛋白（比LFA的3个对数更好）； （3）具有内置连接性和独立的处置墨盒的简单，健壮的仪器。 我们将首先证明适应Daktari病毒蛋白检测测定法的可行性 Zika和登革热抗原。第一阶段将专注于开发和筛选高亲和力和特定抗体 针对zikv和denv的非结构性1（NS1）抗原。然后，我们将结合选定的抗体 磁性和银颗粒，我们将优化测定方案。最后，我们将评估测定 血浆样品的性能。在第二阶段，我们将开发Daktari VIPR IgM测定法，然后适应 VIPR墨盒以实现ZIKV/DENV NS1和IGM分析。我们还将升级Daktari乐器 要合并足够的执行器来执行四个平行测定。最后，集成测试的性能 将在临床样本中进行评估。 凭借简单且敏感的Daktari Zikv/Denv AB/AG组合系统，健康 受过最小培训的工人可以快速准确地诊断世界上任何地方的活跃寨卡病毒感染。