Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material.

Autor:	Stocks BB; Metrology, National Research Council Canada, 1200 Montreal Road, Ottawa, ON K1A 0R6, Canada., Thibeault MP; Metrology, National Research Council Canada, 1200 Montreal Road, Ottawa, ON K1A 0R6, Canada., L'Abbé D; Human Health Therapeutics, National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada., Stuible M; Human Health Therapeutics, National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada., Durocher Y; Human Health Therapeutics, National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada., Melanson JE; Metrology, National Research Council Canada, 1200 Montreal Road, Ottawa, ON K1A 0R6, Canada.
Jazyk:	angličtina
Zdroj:	ACS measurement science au [ACS Meas Sci Au] 2022 Sep 02; Vol. 2 (6), pp. 620-628. Date of Electronic Publication: 2022 Sep 02 (Print Publication: 2022).
DOI:	10.1021/acsmeasuresciau.2c00050
Abstrakt:	Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L -1 ( k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433]. Competing Interests: The authors declare no competing financial interest. (Crown © 2022. Published by American Chemical Society.)
Databáze:	MEDLINE
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