An ELISA and homogeneous assay for serological diagnosis of SARS-CoV2 antibodies

SARS-CoV2 抗体血清学诊断的 ELISA 和同质测定

• 批准号: 10172018
• 负责人: Feifan Yu
• 金额: $ 54.72万
• 依托单位: ALPHATHERA, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-10 至 2021-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172018
• 关键词: 2019-nCoV; Adaptor Signaling Protein; Affinity; Alkaline Phosphatase; Antibodies; Antigens; Binding; Biological Assay; COVID-19; COVID-19 pandemic; Chemicals; Clinical; Collaborations; Complex; Coronavirus; Development; Diagnosis; Diagnostic tests; Disease; Enzyme-Linked Immunosorbent Assay; Enzymes; Goals; Human; Immobilization; Immunity; Immunoassay; Immunoglobulin G; Immunoglobulin M; Infection; Label; Laboratories; Legal patent; Ligation; Methods; Monitor; National Institute of Biomedical Imaging and Bioengineering; Patients; Pennsylvania; Performance; Phase; Post-Translational Protein Processing; Prevalence; Protein Engineering; Proteins; Reagent; Recombinant Proteins; Recombinants; Reporter; Research Personnel; Sampling; Serodiagnoses; Serologic tests; Serological; Serum; Site; Small Business Innovation Research Grant; Speed; Surface; System; Techniques; Technology; Testing; Time; University Hospitals; Viral Antibodies; Viral Antigens; Viral Proteins; Virus; accurate diagnosis; base; combat; comparative; crosslink; density; experience; improved; innovation; novel; pandemic disease; point-of-care diagnostics; response; sortase; vaccine efficacy; vaccine evaluation; workforce needs

The speed and extent of COVID-19 pandemic, caused by the SARS-CoV2 virus, have created unprecedented devastations and challenges. It is now well-established that timely and accurate diagnosis is essential to taming this pandemic. Unlike PCR based tests, which can only diagnose active infections, serological diagnosis of anti- viral antibodies can establish prior infections. Therefore, accurate and efficient serology tests will be important not only for establishing disease prevalence, but also for understanding immunity, testing vaccines efficacy and monitoring disease resurgence. Fortunately, SARS-CoV2 antibodies can be detected using well-established immunoassay formats such as ELISA, ECLIA and CMIA. However, the enormous demand for diagnostic tests is creating unprecedented shortage of otherwise routine reagents and the workforce needed to carry out these tests. Given these headwinds, it will not only be helpful, but in fact crucial, to find potential ways to increase the availability, reduce the complexity and improve the throughput of serological assays in order to meet the projected 5 million daily testing capacity needed by our nation. Our company, in collaboration with our academic partners and with support from NIBIB, has developed and commercialized various novel protein engineering and conjugation technologies that can improve immunoassays. In particular, we have developed more sensitive and robust ELISA and homogenous immunoassays through the use of photoreactive antibody-binding domains (pAbBDs). These small protein adapters can rapidly and site-specifically label antibodies to allow efficient antibody conjugation and immobilization. Since pAbBDs can be produced at large scale, they can also serve as a covalent-binding and highly specific IgG/IgM detecting agent in lieu of secondary antibodies, which are comparatively expensive and complex to produce. Antibodies can be directly labeled with pAbBDs in serum, which eliminates an incubation and wash step required with standard ELISA and significantly reduces assay variability and time. In addition, our previously developed recombinant protein modification technique, termed STEPL (Sortase-Tag Expressed Protein Ligation), will allow recombinant viral antigens to be easily ligated with a chemical tag, and subsequently be site-specifically and covalently immobilized onto a microplate surface, which will lead to improved antibody capture capacity and assay sensitivity. Taken together, these innovations can help both improve existing ELISA- based assays and also enable the creation of novel homogenous serology tests. In collaboration with the Hospital of University of Pennsylvania’s clinical laboratory, we will adapt and rapidly implement our technologies to combat COVID-19 using the methods outlined below: Aim 1. Create “single-wash” SARS-CoV2 ELISA serology assay using pAbBD as IgG/IgM detecting agent; Aim 2. Develop highly sensitive, “no-blocking” and “reusable” SARS-CoV2 ELISA serology assay through site-specific and covalent immobilization of antigens; Aim 3: Develop pAbBD constructs for use in “mix-and-read” homogeneous anti-viral antibody assays.

由SARS-COV2病毒引起的COVID-19的速度和范围已经创造了前所未有的 破坏和挑战。现在已经良好的是，及时，准确的诊断对于驯服至关重要 这个大流行。与基于PCR的测试（只能诊断活跃感染）不同，抗血清学诊断 病毒抗体可以建立先前的感染。因此，准确有效的血清学测试将很重要 不仅用于确定疾病患病率，还用于理解免疫力，测试疫苗效率和 监测疾病复兴。幸运的是，可以使用公认的SARS-COV2抗体检测 免疫测定格式，例如ELISA，ECLIA和CMIA。但是，对诊断测试的巨大需求 正在造成前所未有的常规试剂短缺，以及执行这些试剂所需的劳动力 测试。鉴于这些逆风，不仅会有所帮助，而且实际上至关重要的是寻找增加的潜在方法 可用性，降低复杂性并改善血清学评估的吞吐量，以满足 预计我们国家需要的每日测试能力为500万。 我们的公司与我们的学术合作伙伴合作，并在尼比布的支持下开发和 商业化各种可以改善的新型蛋白质工程和共轭技术 免疫测定。特别是，我们开发了更灵敏，更强大的ELISA和均匀的 通过使用光电反应性抗体结合结构域（PABBD）的免疫测定。这些小蛋白质 适配器可以迅速和特定标记抗体，以允许有效的抗体结合和 固定。由于可以大规模生产PABBD，因此它们也可以用作共价结合和 高度特异性的IgG/IgM检测剂代替了二抗，该抗体相对昂贵，并且 生产复合物。抗体可以在血清中直接用PABBD标记，从而消除孵育 标准ELISA所需的清洗步骤，并大大降低了测定的变异性和时间。另外，我们的 先前开发的重组蛋白质修饰技术称为stepl（表达的sortase-tag 蛋白质结扎），将允许将重组病毒抗原轻松用化学标签连接，然后 将位点特异性和共价固定在微板表面上，这将导致改善的抗体 捕获容量和测定敏感性。综上所述，这些创新可以帮助改善现有的Elisa- 基于基于新型同质血清学检验的基于基于的测定法。与医院合作 在宾夕法尼亚大学的临床实验室中，我们将适应并迅速实施我们的技术 使用以下概述的方法战斗Covid-19 使用PABBD作为IgG/IgM检测剂进行测定；目标2。发展高度敏感，“无障碍”和“可重复使用” SARS-COV2 ELISA血清学测定法，通过抗原特异性和共价固定化；目标3：发展 PABBD构建体用于“混合读”均质抗病毒抗体测定。