A simple protein-based surrogate neutralization assay for SARS-CoV-2
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny H. Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison J. McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny H. Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison J. McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
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Research Article Infectious disease

A simple protein-based surrogate neutralization assay for SARS-CoV-2

Abstract

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.

Authors

Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny H. Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison J. McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras

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Figure 1

Establishment of a surrogate neutralization ELISA (snELISA) to monitor the spike-ACE2 interaction.

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Establishment of a surrogate neutralization ELISA (snELISA) to monitor t...
(A) SARS-CoV-2 attachment to the host cell requires a direct interaction between the host cell receptor, ACE2 (blue), and the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (peach). (B) Ninety-six–well plates are set up in a similar manner for both the detection of anti–SARS-CoV-2 antibodies and neutralizing antibodies. Antigens are adsorbed overnight and incubated with diluted patient serum/plasma samples, monoclonal antibodies, and other affinity reagents. Antigen-specific antibodies are colored black. (C) Principle of direct binding ELISA detection using HRP-conjugated anti–human IgG/A/M. (D) Principle of the snELISA, which uses biotinylated ACE2 for the detection of RBD or spike epitopes that have not been blocked by neutralizing antibodies (using polyHRP-streptavidin). (E) Results of the snELISA using either the RBD or the spike trimer immobilized on the plate (see Supplemental Figures 1 and 2 for the antigen cloning, expression, and purification). The dashed lines are from a sample (CBS39) that was negative for direct RBD/spike binding, while the solid lines are from a positive sample (CBS50; see Supplemental Figure 4A for the direct binding results and Supplemental Table 1 for all OD450 values). (F) snELISA (immobilized RBD) for an expanded set of 4 positive controls with high anti-RBD signals in a single-point direct-binding ELISA (green), 8 negative samples acquired before COVID (red), and 4 samples with low anti-RBD levels (blue; Supplemental Figure 4A). The 5-point curves in E–F were generated from 1 experiment.