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

由SARS-CoV2病毒引起的新冠肺炎大流行的速度和范围已经造成了前所未有的 破坏和挑战。现在公认的是，及时和准确的诊断对于驯服是必不可少的 这场大流行。与只能诊断活动性感染的基于PCR的检测不同，抗-HBs的血清学诊断 病毒抗体可以确定先前的感染情况。因此，准确和有效的血清学检测将是重要的。 不仅用于确定疾病流行率，而且还用于了解免疫力、测试疫苗效力和 监测疾病死灰复燃。幸运的是，SARS-CoV2抗体可以用成熟的 免疫分析形式，如ELISA法、ECLIA和CMIA。然而，对诊断测试的巨大需求 造成了前所未有的常规试剂和进行这些工作所需的劳动力短缺 测试。鉴于这些不利因素，寻找潜在的方法来增加经济增长不仅是有益的，而且实际上至关重要。 可用性，降低复杂性，提高血清学检测的吞吐量，以满足 预计我国每天需要500万个检测能力。 我们公司与我们的学术伙伴合作，并在NIBIB的支持下，开发出了 商业化了各种新的蛋白质工程和接合技术，可以改善 免疫分析。特别是，我们已经开发出更敏感和更强大的酶联免疫吸附试验和同源 通过使用光反应性抗体结合域(PAbBds)进行免疫分析。这些小蛋白质 适配器可以快速和定点标记抗体，以实现有效的抗体结合和 动弹不得。由于pAbBds可以大规模生产，它们也可以作为共价结合和 高特异性免疫球蛋白/免疫球蛋白M检测试剂代替二次抗体，后者相对昂贵和 制作起来很复杂。抗体可以直接在血清中用pAbBds标记，这就省去了孵育 与标准的ELISA法相比，洗涤步骤更简单，大大降低了检测的可变性和时间。此外，我们的 以前开发的重组蛋白修饰技术，称为STEPL(Sortase-Tag Exposed 蛋白质连接)，将允许重组病毒抗原很容易与化学标签连接，并随后 被定位并共价固定在微板表面，这将导致改进的抗体 捕获能力和检测灵敏度。综上所述，这些创新可以帮助改进现有的ELISA- 基于检测，还可以创建新的同质血清学测试。与医院合作 宾夕法尼亚大学临床实验室，我们将调整和迅速实施我们的技术，以 用以下方法对抗新冠肺炎：目的1.建立“一洗”SARS-CoV2ELISA血清学 利用pAbBD作为免疫球蛋白/免疫球蛋白M检测试剂；目的2.开发高灵敏度、无堵塞和可重复使用的方法 通过位点特异性和共价固定抗原的SARS-CoV2 ELISA血清学分析；目标3：发展 PAbBD构建用于“混合读取”的均质抗病毒抗体检测。

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.