DiagnosDisk - a highly sensitive point-of-care test for detecting SARS-CoV-2 antigen in saliva

项目摘要/摘要 SARS-CoV-2是一种全球性的流行病，已在美国造成600多万人感染，18万人死亡 在过去的7个月里，仅在美国。世界卫生组织和美国疾病控制与预防中心都强调需要护理点(POC) 扩大对急性感染的诊断以控制大流行的测试(即隔离感染者)。这个 目前诊断急性SARS-CoV-2感染的金标准是逆转录酶聚合酶 用鼻咽标本进行链式反应(RT-PCR)。这种化验方法非常灵敏，但需要训练有素。 样本采集的人员和配备专门设备的地点。因此，周转时间 从一天到几天不等，延迟隔离、接触者追踪和医疗护理。这些挑战可能 采用侧向流动免疫分析(LFIA)检测自身采集的唾液样本中的病毒抗原。 与鼻咽样本相比，研究表明，唾液样本的分析是 更一致，唾液本身可能含有更高的病毒载量。然而，现有的LFIA用于检测 唾液抗原表现出较低的敏感性(≤40%)，这是由于使用小样本体积和样本造成的 稀释。为实现新冠肺炎疑似病例的及时准确筛查，本项目旨在发展 一种高灵敏度快速检测唾液中SARS-CoV-2抗原的方法 侧写。我们推测，使用≥500ug L唾液作为检测样本，可以显著提高灵敏度 因为更大的样本体积提供了更多的分析物用于检测。新的测试将使用 温度响应型聚合物抗体与诊断盘结合，一种新的流过 化验仪器。该方法在使用丰富的唾液样本进行更一致的分析方面具有创新性。 结果：通过扩大唾液标本体积来加强抗原检测，并采用井检方法。 用温度响应性聚合物试剂建立诊断盘的夹心免疫分析 简化设计，同时最大限度地提高灵敏度。我们将使用高效的聚合物-抗体结合物 分离唾液中的分析物(目标1)并制造诊断磁盘设备以适应更大容量的唾液 提高检测下限的样本(目标2)。新的快速检测的敏感性，它结合了 聚合物-抗体结合物和诊断盘将使用新冠肺炎的唾液样本进行评估 阳性和阴性患者(目标3)。

Project Summary/Abstract SARS-CoV-2, a global pandemic, has caused more than 6 million infections and 180,000 deaths in the United States alone during the past 7 months. Both the WHO and CDC emphasize the need for point-of-care (POC) tests to expand diagnosis of acute infections to control the pandemic (i.e. isolating infected individuals). The current gold standard test for diagnosing acute SARS-CoV-2 infection is reverse transcriptase polymerase chain reaction (RT-PCR) with nasopharyngeal samples. The assay is highly sensitive, but requires well-trained personnel for sample collection and locations with specialized equipment. Therefore, the turnaround time varies from one to few days, delaying isolation, contact tracing, and medical attention. These challenges can be addressed with lateral flow immunoassays (LFIA) to detect viral antigens in self-collected saliva samples. Compared to nasopharyngeal specimens, studies have demonstrated that assays with saliva samples are more consistent and the saliva itself may contain higher viral load. However, existing LFIA for detecting salivary antigens exhibit poor sensitivity (≤ 40%) caused by the use of small sample volumes and sample dilution. To achieve timely and accurate screening of suspected COVID-19 cases, this project aims to develop a rapid assay for detecting SARS-CoV-2 antigens in saliva with high sensitivity to meet WHO's target product profile. We hypothesize that using ≥500 µL saliva as the assay sample can significantly improve the sensitivity because a larger specimen volume provides more analytes for detection. The new test will employ temperature-responsive polymer-antibody conjugates in conjunction with DiagnosDisk, a novel flow-through assay device. The approach is innovative in in using enriched saliva specimens for more consistent assay results, enhancing antigen detection by scaling up the saliva specimen volume, and adopting the well- developed sandwich immunoassay for DiagnosDisk with temperature-responsive polymer reagents that simplify the design while maximizing the sensitivity. We will employ polymer-antibody conjugates that efficiently isolate analytes in saliva (Aim 1) and fabricate the DiagnosDisk device to accommodate larger volume saliva specimens to improve the detection limit (Aim 2). The sensitivity of the new rapid assay, which combines polymer-antibody conjugates and DiagnosDisk, will be evaluated using saliva specimens from COVID-19 positive and negative patients (Aim 3).