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Immunology and Infection

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

Published: June 5th, 2021

DOI:

10.3791/62486

1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

In December 2019, a novel coronavirus was identified, which we now know as SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 is a betacoronavirus belonging to the Coronaviridae family. These enveloped viruses comprise a large positive-sense RNA genome and are responsible for respiratory and intestinal infections in both humans and animals2. As of May 2021 there have been more than 157 million reported cases of COVID-19 globally and more than 3.2 million deaths3. The development of an effective vaccine has become the primary goal of researchers a....

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1. Plating cells (Day 1) for the production and quantification of SARS-CoV-2 pseudovirus

  1. Preparation for tissue culture
    1. Warm 1x Dulbecco's Phosphate-Buffered Saline (DPBS); Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (optional); and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) to 37 °C in a water bath for approximately 15 min.
    2. Disinfect a tissue culture hood with 70% ethanol, and place tissue cultur.......

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This protocol outlines a rapid and effective method for detecting neutralizing antibodies against SARS-CoV-2 S protein via inhibition of VSV-S-eGFP pseudovirus infection (quantifiable by loss of eGFP foci detected). A schematic representation of the protocol is depicted in Figure 1. It is recommended that a commercially available antibody be used as a positive control each time the assay is run to ensure the consistency of the assay. Here, we demonstrate a dilution curve using a commercially.......

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The method described here may be adapted to suit varying lab environments and resources as needed. Importantly, the main limitation of this protocol is the necessity for a containment level 2 space and tissue culture hood. The application of a replicating RNA virus pseudotyped with the SARS-CoV-2 spike, such as VSV-S-eGFP, is a formidable alternative to the SARS-CoV-2 virus, which requires a containment level 3 working area, but may remain a limitation for some groups. All other steps described here are quite flexible an.......

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We would like to thank the Whelan lab for generously providing the VSV-S-eGFP virus used in this protocol (described in Case et al. 2020). We also thank Drs. Bill Cameron and Juthaporn Cowan (and team) for collecting the patient blood samples (REB protocol ID 20200371-01H). The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the generous support from the Ottawa Hospital Foundation and a grant from the Canadian Institutes of Health Research (#448323) and a Fast Grant from the Thistledown foundation for COVID-19 Science to C.S.I. T.R.J. is funded by an Ontario ....

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Name Company Catalog Number Comments
0.25% trypsin-EDTA (Gibco) Fisher scientific LS25200114
ArrayScan VTI HCS Thermo Fisher Scientific Automated fluorescent imager
carboxymethyl cellulose Sigma C5678
Dulbecco's modified Eagle's medium (Gibco) Fisher scientific 10-013-CV
Dulbecco's modified Eagle's medium (Powder) (Gibco) Thermo Fisher Scientific 12-800-017
Dulbecco’s Phosphate-Buffered Saline (DPBS) Fisher scientific 21-031-CV
HEPES Fisher scientific BP-310-500
IgG Isotype Control (mouse) Thermo Fisher Scientific 31903
Penicillin/streptomycin Thermo Fisher Scientific 15070063
SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab SinoBiological 40592-MM57
Vero E6 cells ATCC  CRL-1586

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