Development of Antigen-based Vertical Flow Assay for the Rapid Detection of SARS-CoV-2, Influenza A/B, and RSV

项目摘要 这项第一阶段SBIR计划的目标是开发一种快速、低成本、多重免疫分析方法来检测 常见呼吸道病原体，包括甲型流感(甲型流感)、乙型流感(乙型流感)、呼吸道合胞体 病毒(RSV)和严重急性呼吸综合征冠状病毒-2(SARS-CoV-2)在不到15分钟内完成。这个 这种基于抗原的检测的发展将加快SARS-CoV-2感染的快速诊断，以及 甲型/乙型流感和呼吸道合胞病毒。甲型流感/乙型流感和呼吸道合胞病毒是呼吸道感染的其他常见病原体 比正在进行的SARS-CoV-2大流行更重要。疾控中心的分析表明，可能有10倍 冠状病毒病2019年(新冠肺炎)阳性病例多于报告病例。2这主要是由于 筛选过程缓慢，主要依赖于基于RT-PCR的检测系统。一种快速识别 SARS-CoV-2对于阻止感染的传播至关重要，因为它具有很高的传染性。除了SARS-CoV-2，甲型/乙型流感和呼吸道合胞病毒还会导致相当数量的感染和严重疾病。此外，甲型/乙型流感和呼吸道合胞病毒 感染表现出与SARS-CoV-2感染相似的症状。SARS-CoV-2感染的鉴别 流感或呼吸道合胞病毒感染也是非常必要的，因为如果确诊，流感和呼吸道合胞病毒感染有更好的治疗选择 很早。快速检测将为选择正确的治疗策略和阻止传播提供更好的机会 关于这种疾病的。我们建议开发一种快速、多重检测甲型流感/乙型流感、呼吸道合胞病毒和SARS-CoV-2的方法 不需要任何复杂的仪器。我们的快速检测使用膜上的固定抗体来检测 四种常见呼吸道特异性抗原的捕获及同时定性检测 病原体。我们将使用我们创新设计的顺序注射注射器(SIS)预先填充试剂和 按顺序将它们分散到流动分析试剂盒中。第二抗体等成分 与生物素、洗涤缓冲液、中性亲和素-HRP和具有稳定氢的四甲基联苯胺(TMB)偶联 过氧化氢将被顺序分散到试剂盒中。完成化验后，色斑 该开发人员无需任何特殊仪器即可查看，并可轻松解释。如果需要，阅读器 可用于进一步提高灵敏度和记录测试结果并将其发送给医生的能力。这 可以在不同的护理点(POC)和低资源下在不到15分钟的时间内进行多重分析 无需任何特殊培训的设置。我们将展示我们的测试可以在相同的情况下快速产生结果 使用临床标本对每种病原体进行单链试验的表现水平。我们还将验证我们的 威斯康星医学院合作者实验室的化验方法。我们计划达到100%的特异性 并且比目前的侧向流动分析具有更高的灵敏度。如果开发成功，这种快速、多重测试将被 操作简单，价格低廉，足以满足各种规模的初级保健医生办公室、疗养院、 药房、社区卫生所采用该平台。

Project Summary The goal of this Phase I SBIR proposal is to develop a rapid, low-cost, multiplex immunoassay for the detection of common respiratory tract pathogens, including influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in less than 15 minutes. The development of this antigen-based test will accelerate the rapid diagnosis of SARS-CoV-2 infection, along with influenza A / influenza B and RSV. Influenza A / influenza B and RSV are the other common causative agents of respiratory tract infections other than the ongoing SARS-CoV-2 pandemic. Analyses from the CDC have suggested that there could be 10 times more Coronavirus disease 2019 (COVID-19) positive cases than reported cases. 2 This is primarily due to the slow screening process as it mainly depends on the RT-PCR based testing systems. The rapid identification of SARS-CoV-2 is critical to stop the spread of the infection as it is highly contagious. Apart from SARS-CoV-2, influenza A / influenza B and RSV cause a considerable amount of infections and severe illness. Moreover, influenza A / influenza B and RSV infections show similar symptoms to that of SARS-CoV-2 infection. Differentiating SARS-CoV-2 infections with Flu or RSV is also much needed as Flu and RSV infections have better treatment options if they are diagnosed early. Rapid tests would provide a better opportunity to choose the right treatment strategy and stop the spread of the illness. We propose to develop a rapid, multiplex test for the detection of influenza A / influenza B, RSV, and SARS-CoV-2 without any complex instrumentation. Our rapid test uses immobilized antibodies on the membrane for the capturing and simultaneous qualitative detection of specific antigens for four common respiratory tract pathogens. We will use our innovatively designed sequential injection syringe (SIS) to pre-fill the reagents and disperse them sequentially into the flow-through assay cassette. The components such as the 2nd antibody conjugated with biotin, wash buffers, neutravidin-HRP, and tetramethylbenzidine (TMB) with stable hydrogen peroxide will be sequentially dispersed into the assay cassette. After completing the assay, the colored spots that develop can be viewed without any special instrumentation and can be interpreted easily. If needed, a reader can be used to further increase the sensitivity and ability to record and send test result to the physicians. This multiplex assay can be performed in less than 15 minutes in various point-of-care (POC) and low resource settings without any special training. We will demonstrate that our test can deliver rapid results with the same level of performance as singleplex tests for each pathogen using clinical specimens. We will also validate our assay method at our collaborator's lab at the Medical College of Wisconsin. We plan to achieve 100% specificity and higher sensitivity than current lateral flow assays. If successfully developed, this rapid, multiplex test will be simple to perform and inexpensive enough for all sizes of primary-care physician's offices, nursing homes, pharmacies, and community health clinics to adopt the platform.