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

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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

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Most cited references 12

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A Novel Coronavirus from Patients with Pneumonia in China, 2019

Na Zhu, Dingyu Zhang, Wenling Wang … (2020)

Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.)

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A new coronavirus associated with human respiratory disease in China

Fan Wu, Su Zhao, Bin Yu … (2020)

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health 1–3 . Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing 4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China 5 . This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

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Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Daniel Wrapp, Nianshuang Wang, Kizzmekia S. Corbett … (2020)

Structure of the nCoV trimeric spike The World Health Organization has declared the outbreak of a novel coronavirus (2019-nCoV) to be a public health emergency of international concern. The virus binds to host cells through its trimeric spike glycoprotein, making this protein a key target for potential therapies and diagnostics. Wrapp et al. determined a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy. Using biophysical assays, the authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor. They also tested three antibodies known to bind to the SARS-CoV spike protein but did not detect binding to the 2019-nCoV spike protein. These studies provide valuable information to guide the development of medical counter-measures for 2019-nCoV. Science, this issue p. 1260

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Author and article information

Contributors

Florian Krammer: florian.krammer@mssm.edu

Journal

Journal ID (nlm-ta): Curr Protoc Microbiol

Journal ID (iso-abbrev): Curr Protoc Microbiol

Journal ID (doi): 10.1002/(ISSN)1934-8533

Journal ID (publisher-id): CPMC

Title: Current Protocols in Microbiology

Publisher: John Wiley and Sons Inc. (Hoboken )

ISSN (Print): 1934-8525

ISSN (Electronic): 1934-8533

Publication date (Electronic): 17 April 2020

Publication date (Print): June 2020

Volume: 57

Issue: 1 ( doiID: 10.1002/cpmc.v57.1 )

Electronic Location Identifier: e100

Affiliations

[ ¹ ] Department of Microbiology Icahn School of Medicine at Mount Sinai New York New York

[ ² ] Graduate School of Biomedical Sciences Icahn School of Medicine at Mount Sinai New York New York

[ ³ ] Department of Biotechnology University of Natural Resources and Life Sciences Vienna Austria

[ ⁴ ] Global Health Emerging Pathogens Institute Icahn School of Medicine at Mount Sinai New York

[ ⁵ ] Division of Infectious Diseases, Department of Medicine Icahn School of Medicine at Mount Sinai New York New York

Author notes

[*] [* ]Corresponding author: florian.krammer@ 123456mssm.edu

[†]

These authors contributed equally to this work

Article

Publisher ID: CPMC100

DOI: 10.1002/cpmc.100

PMC ID: 7235504

PubMed ID: 32302069

SO-VID: a01411d0-6aa9-4f38-843f-0ee5fda49b1e

License:

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Page count

Figures: 4, Tables: 0, Pages: 15, Words: 6569

Funding

Funded by: NIAID , open-funder-registry 10.13039/100000060;

Award ID: HHSN272201400008C

Custom metadata

source-schema-version-number 2.0

cover-date June 2020

details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.2 mode:remove_FC converted:19.05.2020

Keywords: covid19,covid‐19,elisa,protein expression,sars‐cov‐2,serological assay

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Keywords: covid19, covid‐19, elisa, protein expression, sars‐cov‐2, serological assay

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

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Novel Coronavirus Disease COVID-19

Most cited references 12

A Novel Coronavirus from Patients with Pneumonia in China, 2019

A new coronavirus associated with human respiratory disease in China

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

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