SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup

Current Protocols in Microbiology Pub Date : 2020-04-17 DOI:10.1002/cpmc.100

Daniel Stadlbauer, Fatima Amanat, Veronika Chromikova, Kaijun Jiang, Shirin Strohmeier, Guha Asthagiri Arunkumar, Jessica Tan, Disha Bhavsar, Christina Capuano, Ericka Kirkpatrick, Philip Meade, Ruhi Nichalle Brito, Catherine Teo, Meagan McMahon, Viviana Simon, Florian Krammer

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引用次数: 608

Abstract

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. Current Protocols in Microbiology published by Wiley Periodicals LLC.

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

Abstract Image

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SARS-CoV-2在人体内的血清转化:血清学测定、抗原产生和测试设置的详细方案

2019年底，中国发现了非典型肺炎病例。病原很快被确定为一种冠状病毒(命名为SARS-CoV-2)，此后引发了一场大流行。已经建立了几种特定检测病毒核酸的方法，但这些方法只能在短时间内检测病毒，通常是在急性感染期间。迫切需要进行血清学分析，以开展血清调查，了解针对该病毒产生的抗体反应，并确定对再次感染具有潜在免疫力的个体。在这里，我们描述了一种详细的方案，用于表达源自SARS-CoV-2刺突蛋白的抗原，该抗原可作为免疫测定的底物，以及两阶段血清学酶联免疫吸附测定(ELISA)。这些测定法可用于研究和临床实验室的检测。©2020作者。Wiley期刊有限责任公司发表的现行微生物学方案基本方案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品中与SARS-CoV-2刺突蛋白结合的抗体的两阶段ELISA

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来源期刊

Current Protocols in Microbiology Immunology and Microbiology-Parasitology

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期刊介绍： Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.