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-CoV 2病毒引起的COVID-19大流行的速度和范围, 挑战与挑战。现在已经公认,及时准确的诊断对驯服 这种流行病。与只能诊断活动性感染的基于PCR的测试不同,抗- 病毒抗体可以确定先前的感染。因此,准确和有效的血清学测试将是重要的 不仅用于确定疾病流行率,还用于了解免疫力,测试疫苗效力, 监测疾病复发。幸运的是,SARS-CoV 2抗体可以使用成熟的 免疫测定形式如ELISA、ECLIA和CMIA。然而,对诊断测试的巨大需求 正在造成前所未有的短缺,否则常规试剂和劳动力需要进行这些 试验.考虑到这些不利因素,找到潜在的方法来增加 可用性,降低复杂性并提高血清学测定的通量,以满足 预计我们国家每天需要500万个检测能力。 我们公司与我们的学术合作伙伴合作,并在NIBIB的支持下, 商业化的各种新的蛋白质工程和缀合技术,可以改善 免疫测定。特别是,我们已经开发了更灵敏和更强大的ELISA和同质化。 通过使用光反应性抗体结合结构域(pAbBD)进行免疫测定。这些小蛋白质 衔接子可以快速和位点特异性地标记抗体以允许有效的抗体缀合, 固定化。由于pAbBD可以大规模生产,因此它们也可以用作共价结合和免疫抑制剂。 高特异性IgG/IgM检测试剂代替相对昂贵且 复杂的生产。抗体可以在血清中直接用pAbBD标记,这消除了孵育 和标准ELISA所需的洗涤步骤,并显著减少测定变异性和时间。另外我们 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容易地与化学标签连接,并且随后 位点特异性和共价固定在微孔板表面上,这将导致改进的抗体 捕获能力和测定灵敏度。总之,这些创新可以帮助改善现有的ELISA- 并且还能够创建新的同质血清学测试。与医院合作 宾夕法尼亚大学的临床实验室,我们将适应并迅速实施我们的技术, 使用以下方法抗击COVID-19:目标1.创建“一次洗涤”SARS-CoV 2 ELISA血清学 以pAbBD为IgG/IgM检测试剂进行检测;目的2.开发高灵敏度、“无阻塞”和“可重复使用”的 SARS-CoV 2 ELISA血清学检测方法的建立 pAbBD构建体用于“混合-读取”均相抗病毒抗体测定。

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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