ABSTRACT
Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level.The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure;this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability.Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins.To reach the main text of this paper, click on Final Report.
ABSTRACT
The year 2020 will be remembered for the most pernicious epidemic of modern history. The SARS-CoV-2 coronavirus has affected millions of people worldwide and has triggered an unprecedented race for the development of diagnostic tests and therapeutical vaccines. These technologies require befitting measurements that underpin reliability and patient safety. The level of blood-borne antibodies against SARS-CoV-2 is correlated initially with the extent of infection and subsequently with the degree of immunity attained. This renders their accurate measurement an important target for immunometric and liquid chromatography-mass spectrometry methods. Suitable reference materials and methods are being developed to ensure the reliability and consistency of such measurements. Solutions of purified monoclonal IgG may prove useful as primary calibrator materials for the development of calibration hierarchies for the measurement of blood-borne antibodies against SARS-CoV-2. Bottom-up approaches can be applied to the quantification of such structurally complex large molecules. These consist of trypsin and Lys C digestion and quantification of the resulting proteotypic peptides as surrogate analytes for an IgG monoclonal antibody. Establishing the metrological traceability of this procedure requires peptides whose purity has been determined accurately, for example with quantitative nuclear magnetic resonance (qNMR). The H-1 qNMR method allows for the mass fraction assignment of peptides by accurate quantification of H-1 resonance signals specific for both the peptide and for a certified reference material used as internal standard in the peptide solution. The current paper describes the qNMR characterization of five peptides that could be used in a double isotope dilution method for the quantification of SARS-CoV-2 IgG monoclonal antibodies in solution.