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

项目总结/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（COVID-19）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。传播速度快，部分个体渐进感染，目前 许多国家的疫苗供应仍然有限，而不断的病毒变异使COVID-19成为一种 对人类健康和经济构成前所未有的全球性威胁。中和抗体（nAb）识别SARS- CoV-2的刺突蛋白和阻断细胞进入，作为免疫系统中的第一反应者， 病原体清除评估nAb对病毒病原体的有效性对于理解 自然感染后或接种疫苗后的绝育免疫水平和持续时间，特别是考虑到 具有疫苗逃逸潜力的新型变体的增加。然而，许多可用的中和测定用于 评估nAb功能涉及病毒的繁殖，因此需要在生物安全条件下进行此类测定。 3级（BSL 3）实验室设置，不幸的是，这是许多研究人员或临床医生不可用的。此外，本发明还提供了一种方法， 基于临床实验室的抗体测试测量响应SARS-CoV-2抗原的总Ab水平， 功能性评估病原体结合的Ab，因此不能预测中和活性。弥补这些 技术差距，我们提出了一个多学科的研究计划，以解决低， 成本，高通量，快速，简单和定量分析格式在研究COVID-19免疫反应。的 亚利桑那州立大学的研究人员在纳米传感器设计，抗体表征和 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 纳米颗粒（MNP）为基础的nAb测定平台。该平台提供了几个关键特性， 以前的技术。首先，MNP测定是定量的和准确的，具有预期的动态范围： 3至4 log，检测限在皮摩尔范围内。此外，该测定可以快速实施。 检测格式，无需任何洗涤步骤，因此显著简化了其操作， 几分钟，并使其可行的大规模测试。重要的是，该检测方法能够检测病毒变体 通过靶向结合刺突蛋白上对突变敏感的表位。此外， 可以在与高通量筛选兼容的孔板中进行， 组件，使系统在检测和数据分析方面实现自动化。我们设想， 这种快速和定量的nAb检测也将有助于及时确定恢复期药物的潜在最佳用途。 血浆和抗体治疗与未来出现的新病毒。其成本低、操作简单、自动化程度高 这种能力在与COVID-19感染相关的免疫反应的纵向研究中也非常有用， 疫苗接种，以及由于突变而导致的潜在病毒逃逸。nAb传感也可用于大群体 血清监测，以确定针对任何病毒株的群体免疫力（群体免疫力）水平。

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.