Development of rapid, low-cost, and high throughput COVID-19 antibody assays

摘要 数百万人通过感染或接种疫苗产生了新冠肺炎抗体，这一数字将继续保持下去 来增加。然而，抗体(通过感染或疫苗)迅速衰退，并因个体而异。上一首 研究表明，由于抗体水平降低，两者结合在一起，保护效果降低。 随着弱毒株的出现，德尔塔变种(和其他变种)最近在全球范围内广泛传播。第三剂 美国疾病控制与预防中心现在推荐使用疫苗。定期监测新冠肺炎中和抗体可以 确定需要加强疫苗的个人，告知发病率研究，并指导有关政策 为人群接种疫苗的频率。虽然侧向流动分析有可能用于快速COVID- 19中和抗体检测，它们不能提供准确的中和抗体水平，而且有很大的 变异，使它们成为跟踪和预测未来感染易感性的糟糕工具。更准确 在集中式实验室进行抗体测试需要2-3天的时间，而且成本很高(150美元)。 OptoBio开发了一种通用微流控96孔酶标板(MicroFluere®)技术， 将分析时间缩短5-10倍，将样品和试剂消耗减少5-6倍，并增加动态 与传统的96孔酶标板相比，范围扩大了10倍。第一阶段研究的目标是 演示利用MicroFluere®开发廉价(10美元)、快速(20分钟)和 高通量(240次/小时)检测不同变异株的新冠肺炎中和抗体。目前没有 存在能够以高通量和低成本对中和抗体进行量化的产品 大规模筛查。第一阶段有两个具体目标。 目的#1.设计、测试和优化6种类型新冠肺炎的中和抗体检测方法。我们 将首先为所有6种SARS-CoV-2变种选择内部标准。然后我们会优化化验方法 如平板表面涂层、样品稀释系数和孵化时间等参数。最后，我们会 评估分析性能，如回收率和板对板的重复性。 目的#2.用人体血液样本和基准验证适用性。该检测方法是在 AIM#1将在OptoBio的BSL-2实验室使用75个商业购买的人类血清样本进行测试。五 其他病毒感染也将包括在内，以进行交叉反应评估。所有的结果都将与 用市面上出售的SARS-CoV-2中和抗体试剂盒获得的结果。 在第二阶段，OptoBio将检测更多的样本和添加更多的变种，完成高通量的自动化 系统，开发具有足够保质期的预涂覆MicroFluere®平板，并开发使用更多 方便的基质，如指刺全血。我们设想的产品将满足未满足的需求 在临床实验室和护理点检测站点进行快速、准确和低成本的新冠肺炎抗体检测。这个 同样的产品和技术也可以适用于可能发生的其他病毒大流行。

ABSTRACT Millions have developed COVID-19 antibodies through infection or vaccines, and this number will continue to increase. However, antibodies (via infection or vaccines) decay rapidly and vary among individuals. Previous studies have shown decreased protection efficacy because of reduced antibody levels, which, in combination with muted strains, has led to recent wide spread of Delta variant (and other variants) worldwide. A third dose of vaccine is now recommended by the CDC. Regular monitoring of COVID-19 neutralizing antibodies can identify individuals requiring booster vaccines, inform incidence studies, and guide policies regarding vaccination frequency for the population. Although lateral flow assays can potentially be used for rapid COVID- 19 neutralizing antibody tests, they do not provide an accurate level of neutralizing antibodies and has large variations, making them a poor tool to track and predict susceptibility to future infections. More accurate antibody tests done at centralized labs take 2-3 days to turnaround and have a high cost ($150). OptoBio has developed a general purpose microfluidic 96-well ELISA plate (MicroFluere®) technology that reduces assay time 5-10 fold, reduces sample and reagent consumption 5-6 fold, and increases dynamic range by up to 10 fold as compared to traditional 96-well ELISA plate. The goal of Phase I study is to demonstrate the feasibility of leveraging MicroFluere® to develop an inexpensive (<$10), rapid (20 min), and high throughput (240 tests/hour) assay for COVID-19 neutralizing antibodies of different variants. Currently no product exists that is able to quantify neutralizing antibody at a high throughput and low cost at point-of-care for mass screening. There are two specific aims in Phase 1. Aim #1. Design, test, and optimize neutralizing antibody assays for 6 types of COVID-19 strains. We will first select internal standards for all 6 types of SARS-CoV-2 variants. Then we will optimize the assay parameters such as the plate surface coating, sample dilution factors, and incubation time. Finally, we will evaluate the assay performance such as recovery rate and plate-to-plate repeatability. Aim #2. Validate applicability with human blood samples and benchmark. The assay developed in Aim #1 will be tested at a BSL-2 lab at OptoBio using 75 human serum samples purchased commercially. Five other virus infections will also be included for cross-reactivity evaluation. All the results will be compared with those obtained with SARS-CoV-2 neutralizing antibody kits commercially available. In Phase II, OptoBio will test more samples and add more variants, complete a high throughput automated system, develop pre-coated MicroFluere® plates with adequate shelf lifetime, and develop assays using more convenient matrices such as finger-prick whole blood. Our envisioned product will address the unmet need for rapid, accurate, and low cost COVID-19 antibody detection at clinical labs and point-of-care testing sites. The same product and technology can also be adapted for other viral pandemics that may occur.