Current Protocols in Immunology
PROTOCOL

Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies

Abigail Vanderheiden

Abigail Vanderheiden

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Yerkes National Primate Research Center, Atlanta, Georgia

These authors contributed equally to this work.

Contribution: Conceptualization, Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing

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Venkata Viswanadh Edara

Venkata Viswanadh Edara

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Yerkes National Primate Research Center, Atlanta, Georgia

These authors contributed equally to this work.

Contribution: Conceptualization, Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing

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Katharine Floyd

Katharine Floyd

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Yerkes National Primate Research Center, Atlanta, Georgia

Contribution: Conceptualization, Data curation, Methodology, Writing - review & editing

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Robert C. Kauffman

Robert C. Kauffman

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

Contribution: Conceptualization, Resources

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Grace Mantus

Grace Mantus

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

Contribution: Conceptualization, Data curation, Resources

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Evan Anderson

Evan Anderson

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Contribution: Conceptualization, Data curation, Resources, Writing - review & editing

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Nadine Rouphael

Nadine Rouphael

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Department of Medicine, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Contribution: Conceptualization, Resources

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Sri Edupuganti

Sri Edupuganti

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Department of Medicine, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Contribution: Resources

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Pei-Yong Shi

Pei-Yong Shi

Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas

Contribution: Methodology, Resources

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Vineet D. Menachery

Vineet D. Menachery

Department of Microbiology and Immunology, Institute for Human Infection and Immunity, World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, Texas

Contribution: Methodology, Resources

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Jens Wrammert

Jens Wrammert

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

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Mehul S. Suthar

Corresponding Author

Mehul S. Suthar

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia

Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia

Yerkes National Primate Research Center, Atlanta, Georgia

Corresponding author: mehul.s.suthar@emory.edu

Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, ​Investigation, Supervision, Visualization, Writing - review & editing

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First published: 20 November 2020
Citations: 43
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Abstract

SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone−derived SARS-CoV-2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high-throughput setting, and are compatible with large-scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC

Basic Protocol: Focus reduction neutralization test

Alternate Protocol: mNeonGreen-based focus reduction neutralization test (FRNT-mNG)