Stadlbauer Daniel, Amanat Fatima, Chromikova Veronika, Jiang Kaijun, Strohmeier Shirin, Arunkumar Guha Asthagiri, Tan Jessica, Bhavsar Disha, Capuano Christina, Kirkpatrick Ericka, Meade Philip, Brito Ruhi Nichalle, Teo Catherine, McMahon Meagan, Simon Viviana, Krammer Florian
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York.
Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.
Curr Protoc Microbiol. 2020 Jun;57(1):e100. doi: 10.1002/cpmc.100.
In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.
2019年末，中国检测到非典型肺炎病例。病原体很快被确定为一种β冠状病毒（命名为SARS-CoV-2），自那时起引发了一场大流行。已经建立了几种能够特异性检测病毒核酸的方法，但这些方法仅能在短时间内检测到病毒，通常是在急性感染期间。迫切需要血清学检测来进行血清学调查，了解机体针对该病毒产生的抗体反应，并识别可能对再次感染具有免疫力的个体。在此，我们描述了一种详细方案，用于表达源自SARS-CoV-2刺突蛋白的抗原，该抗原可作为免疫检测的底物，以及一种两阶段血清学酶联免疫吸附测定（ELISA）。这些检测可用于研究以及临床实验室检测。© 2020作者。基本方案1：哺乳动物细胞转染和蛋白质纯化 基本方案2：用于高通量筛选人血清样本中与SARS-CoV-2刺突蛋白结合抗体的两阶段ELISA。
