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

基本信息

  • 批准号:
    10270691
  • 负责人:
  • 金额:
    $ 11.96万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2017
  • 资助国家:
    美国
  • 起止时间:
    2017-04-01 至 2021-08-09
  • 项目状态:
    已结题

项目摘要

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 大流行的速度和范围造成了前所未有的 破坏和挑战。现在已经明确,及时、准确的诊断对于驯服疾病至关重要 这次大流行。与基于 PCR 的检测只能诊断活动性感染不同,抗病毒的血清学诊断 病毒抗体可以建立先前的感染。因此,准确、高效的血清学检测非常重要 不仅可以用于确定疾病流行情况,还可以用于了解免疫力、测试疫苗功效和 监测疾病复发。幸运的是,可以使用成熟的方法检测 SARS-CoV2 抗体 免疫测定形式,例如 ELISA、ECLIA 和 CMIA。然而,诊断测试的巨大需求 正在造成常规试剂和执行这些操作所需的劳动力的前所未有的短缺 测试。考虑到这些不利因素,寻找潜在的方法来增加 可用性,降低复杂性并提高血清学检测的通量,以满足 预计我们国家每天需要500万次检测能力。 我们公司与我们的学术合作伙伴合作并在 NIBIB 的支持下,开发并 将各种新型蛋白质工程和缀合技术商业化,可以改善 免疫测定。特别是,我们开发了更灵敏、更稳健的 ELISA 和同质 通过使用光反应性抗体结合域 (pAbBD) 进行免疫测定。这些小蛋白质 接头可以快速地、位点特异性地标记抗体,以实现有效的抗体缀合和 固定化。由于 pAbBD 可以大规模生产,因此它们也可以作为共价结合和 高特异性 IgG/IgM 检测剂代替二抗,二抗相对昂贵且 生产复杂。抗体可以直接用血清中的 pAbBD 进行标记,从而消除了孵育 标准 ELISA 所需的洗涤步骤可显着减少测定变异性和时间。此外,我们的 先前开发的重组蛋白修饰技术,称为STEPL(Sortase-Tag Expressed 蛋白质连接),将允许重组病毒抗原轻松地与化学标签连接,然后 被位点特异性地共价固定在微孔板表面上,这将导致抗体的改进 捕获能力和测定灵敏度。总而言之,这些创新可以帮助改进现有的 ELISA- 基于测定,还能够创建新颖的同质血清学测试。与医院合作 宾夕法尼亚大学临床实验室,我们将调整并快速实施我们的技术 使用下述方法对抗 COVID-19: 目标 1. 创建“单次清洗”SARS-CoV2 ELISA 血清学 使用pAbBD作为IgG/IgM检测剂进行测定;目标2.开发高灵敏、“无阻塞”、“可重用” 通过抗原的位点特异性和共价固定进行 SARS-CoV2 ELISA 血清学测定;目标 3:发展 pAbBD 构建体用于“混合读取”均质抗病毒抗体测定。

项目成果

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Feifan Yu其他文献

Feifan Yu的其他文献

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{{ truncateString('Feifan Yu', 18)}}的其他基金

TABA: Tools for the site-specific labeling and immobilization of antibodies for immunoassays
TABA:用于免疫测定的抗体位点特异性标记和固定化工具
  • 批准号:
    10408659
  • 财政年份:
    2021
  • 资助金额:
    $ 11.96万
  • 项目类别:
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
  • 财政年份:
    2020
  • 资助金额:
    $ 11.96万
  • 项目类别:
TRIM21-mediated degradation of antibody-targeted cytosolic proteins
TRIM21 介导的抗体靶向胞浆蛋白降解
  • 批准号:
    10006659
  • 财政年份:
    2020
  • 资助金额:
    $ 11.96万
  • 项目类别:
Generation of antibody-drug conjugates by proximity-based sortase-mediated ligation
通过基于邻近的分选酶介导的连接生成抗体-药物缀合物
  • 批准号:
    10437944
  • 财政年份:
    2017
  • 资助金额:
    $ 11.96万
  • 项目类别:
Generation of antibody-drug conjugates by proximity-based sortase-mediated ligation
通过基于邻近的分选酶介导的连接生成抗体-药物缀合物
  • 批准号:
    10323853
  • 财政年份:
    2017
  • 资助金额:
    $ 11.96万
  • 项目类别:
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