1. Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials.
- Author
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Stocks BB, Thibeault MP, L'Abbé D, Umer M, Liu Y, Stuible M, Durocher Y, and Melanson JE
- Subjects
- Humans, Biotinylation, COVID-19 virology, COVID-19 Serological Testing methods, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus metabolism, Angiotensin-Converting Enzyme 2 metabolism, Angiotensin-Converting Enzyme 2 chemistry, SARS-CoV-2, Reference Standards
- Abstract
Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L
-1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L-1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values., (© 2024. Crown.) more...- Published
- 2024
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