Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Bioinformatics-Based Design Strategy and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein

Partha Ray
ORCID logo ,
Melissa Ledgerwood-Lee
,
Howard Brickner
,
Alex E. Clark
,
Aaron Garretson
,
Rishi Graham
,
Westley Van-Zant
,
Aaron F. Carlin
ORCID logo ,
Eliah S. Aronoff-Spencer
* ORCID logo
Version 1 : Received: 30 March 2024 / Approved: 1 April 2024 / Online: 1 April 2024 (17:21:17 CEST)

A peer-reviewed article of this Preprint also exists.

Ray, P.; Ledgerwood-Lee, M.; Brickner, H.; Clark, A.E.; Garretson, A.; Graham, R.; Van Zant, W.; Carlin, A.F.; Aronoff-Spencer, E.S. Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels. Viruses 2024, 16, 662. Ray, P.; Ledgerwood-Lee, M.; Brickner, H.; Clark, A.E.; Garretson, A.; Graham, R.; Van Zant, W.; Carlin, A.F.; Aronoff-Spencer, E.S. Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels. Viruses 2024, 16, 662.

Abstract

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for calibration and quality assurance of inactivated SARS-COV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. Heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)- C-terminal domain (CTD) across all SARS-COV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprising of UV-inactivated SARS CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (Limit of Detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a twenty amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust ‘pan-variant’ antigen-based assays for viruses prone to high mutational rates.

Keywords

SARS-CoV-2; Nucleocapsid protein; Monoclonal and Polyclonal Antibodies; Enzyme-Linked Immunoassay; Peptide epitope mapping; Viral Quality Assurance; RADx; COVID-19 diagnostics

Subject

Biology and Life Sciences, Virology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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