High-Throughput, Rapid, and Epitope-Specific Quantification of Neutralizing Antibodies Using Digital Nanoparticle Sensors

项目摘要/摘要 截至2021年6月中旬，由SARS-CoV-2病毒引起的新型冠状病毒病(新冠肺炎)已感染 约1.8亿人，造成全球约380万人死亡。确诊病例超过3400万例 仅在美国就有62万人死亡。在一些个体中的快速传播，渐近感染，目前 在许多国家，疫苗供应仍然有限，加上病毒不断变异，使得新冠肺炎成为 对人类健康和经济的前所未有的全球威胁。中和抗体(NAB)识别SARS- 冠状病毒2型S刺激蛋白并阻断细胞进入，在免疫系统中起到第一反应 病原体清除。评估nabs对病毒病原体的有效性对于理解 自然感染或接种疫苗后的灭菌免疫水平和持续时间，尤指考虑到 具有疫苗逃逸潜力的新变种的兴起。然而，许多可用的中和试验用于 评估NAB功能涉及病毒的传播，因此需要在生物安全的情况下进行此类检测 3级(BSL3)实验室设置，不幸的是，这对许多研究人员或临床医生来说是不可用的。此外, 基于临床实验室的抗体检测测量对SARS-CoV-2抗原反应的总抗体水平，而不是 功能评估病原体结合抗体，因此不能预测中和活性。为了弥合这些 技术差距，我们提出了一个多学科的研究计划，以解决低- 研究新冠肺炎免疫应答的成本、高通量、快速、简单和定量的分析模式。这个 亚利桑那州立大学的研究人员在纳米传感器设计、抗体特性和 冠状病毒将合作开发一种新的高回报研究策略，以建立一种金属 基于纳米颗粒(MNP)的NAB分析平台。该平台提供了几个关键功能，区别于 以前的技术。首先，MNP分析是定量和准确的，预期动态范围为 3~4个对数，检出限在皮摩尔范围内。此外，该检测方法可以快速实现 检测格式，无需任何洗涤步骤，从而大大简化了操作，将分析时间减少到 几分钟，并使其可以进行大规模测试。重要的是，这种检测方法能够检测出病毒的变种 通过靶向结合对突变敏感的尖峰蛋白上的表位。此外，读数 可在与高通量筛选兼容的井板中进行，并添加便携式电子设备 组件，使系统在检测和数据分析方面实现自动化。我们设想的是就业 这种快速和定量的NAB检测也将有助于及时确定恢复期的潜在最佳用途 用未来出现的新病毒治疗血浆和抗体。其成本低、操作简单、自动化程度高 这种能力在对新冠肺炎感染相关免疫反应的纵向研究中也非常有用， 疫苗接种，以及由于突变可能导致的病毒逃逸。NAB感知也可以用于大人口 在确定针对任何病毒株的基于人群的免疫(群体免疫)水平方面进行血清监测。

Project Summary/Abstract As of mid June 2021, the new coronavirus disease (COVID-19), caused by SARS-CoV-2 virus, has infected ~180 million people and causing ~3.8 million deaths globally. There are more than 34 million confirmed cases and ~620,000 deaths in the U.S alone. The fast transmission, asymptotic infection in some individuals, currently still limited supplies of vaccines in many countries, and constant virus mutation have made COVID-19 an unprecedented global threat to human health and economics. Neutralizing antibodies (nAbs) recognize SARS- CoV-2’s spike protein and block cellular entry, acting as the first responders in the immune system towards pathogen clearance. Evaluating the effectiveness of nAbs against viral pathogens is important in understanding the level and duration of sterilizing immunity after natural infection or following vaccination, particularly given the rise of novel variants with vaccine escape potential. However, many of the available neutralizing assays used to assess nAb function involve propagation of viruses and thus require such assays be conducted in a biosafety level 3 (BSL3) lab settings, which, unfortunately, is unavailable to many researchers or clinicians. In addition, clinical laboratory-based antibody tests measure the total Ab level responding to SARS-CoV-2 antigens, without functionally evaluating pathogen-bound Abs and therefore cannot predict neutralizing activity. To bridge these technological gaps, we propose a multidisciplinary research plan to address the fundamental challenges in low- cost, high-throughput, fast, simple, and quantitative assay format in studying COVID-19 immune response. The investigators at ASU with complementary expertise in nanosensor design, antibody characterization, and coronaviruses will collaboratively develop a new and high-reward research strategy to establish a metal nanoparticle (MNP)-based nAb assay platform. This platform presents a few key features distinguishing it from previous technologies. First, the MNP assays are quantitative and accurate, with an expected dynamic range of 3 to 4 logs and a detection limit in the picomolar range. Further, this assay can be implemented in a rapid detection format without any washing steps, thus significantly simplifying its operation, reducing assay time to a few minutes, and making it feasible for mass-testing. Importantly, the assay is capable of detecting virus variants by targeted binding to the epitopes on the spike protein that are sensitive to mutations. Additionally, the readout can be performed in a well plate compatible with high-throughput screening with added portable electronic components, making the system automated in both detection and data analysis. We envision the employment of this rapid and quantitative nAb assay will also help timely determine the potential best uses of convalescent plasma and antibody treatment with future emerging novel viruses. Its low cost, simple operation, and automation capability are also very useful in longitudinal studies of the immune response related to COVID-19 infection, vaccination, and potential viral escape due to mutations. The nAb sensing can also be used for large-population sero-surveillance in determining the level of population-based immunity (herd immunity) against any virus strains.