Abstract
Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.
Highlights
The recently emerged SARS-CoV-2 coronavirus is the causative agent of the current COVID-19 pandemic which has resulted in nearly 300 million infections and over five million deaths at the time of this report [1]
The recent development of a SARS-CoV-2 nucleic acid reference material allows for enhanced reliability of molecular diagnostics [11], yet to date, protein reference materials are lacking. To address this current lack of standardization in COVID-19 rapid testing, we have developed a SARS-CoV-2 spike protein reference material, SMT1-1 [12]
Solution-phase reference materials are often highly homogeneous; inhomogeneity can occur throughout the production process for a variety of reasons, i.e., changing environmental conditions, interaction between the sample and its container, and contamination
Summary
The recently emerged SARS-CoV-2 coronavirus is the causative agent of the current COVID-19 pandemic which has resulted in nearly 300 million infections and over five million deaths at the time of this report [1]. Keywords COVID-19 · SARS-CoV-2 · Spike protein · Reference materials · Isotope dilution To address this current lack of standardization in COVID-19 rapid testing, we have developed a SARS-CoV-2 spike protein reference material, SMT1-1 [12].
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