Flow Cytometry-Based Measurement of Antibodies Specific for Cell Surface-Expressed Folded SARS-CoV-2 Receptor-Binding Domains
Author:
Sehgal Al Nasar Ahmed1, Safran Jera1, Kratzer Bernhard1ORCID, Gattinger Pia2ORCID, Stieger Robert B.1, Musiejovsky Laszlo1, Trapin Doris1, Ettel Paul1, Körmöczi Ulrike1, Rottal Arno1, Borochova Kristina2, Dorofeeva Yulia2, Tulaeva Inna23, Weber Milena2, Grabmeier-Pfistershammer Katharina1ORCID, Perkmann Thomas4, Wiedermann Ursula5, Valenta Rudolf2367, Pickl Winfried F.17
Affiliation:
1. Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 2. Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 3. Laboratory for Immunopathology, Department of Clinical Immunology and Allergology, Sechenov First Moscow State Medical University, 119991 Moscow, Russia 4. Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria 5. Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 6. NRC Institute of Immunology FMBA of Russia, 115478 Moscow, Russia 7. Karl Landsteiner University of Health Sciences, 3500 Krems, Austria
Abstract
Background: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. Methods: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. Results: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. Conclusion: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.
Funder
Danube Allergy Research Cluster Government of Lower Austria Medical University of Vienna
Reference62 articles.
1. The origins and potential future of SARS-CoV-2 variants of concern in the evolving COVID-19 pandemic;Otto;Curr. Biol.,2021 2. Kumar, D., Roy, S.S., Rastogi, R., Arora, K., Undale, A., Gupta, R., Arora, N.M., and Kundu, P.K. (2023). VLP-ELISA for the Detection of IgG Antibodies against Spike, Envelope, and Membrane Antigens of SARS-CoV-2 in Indian Population. Vaccines, 11. 3. Gattinger, P., Ohradanova-Repic, A., and Valenta, R. (2023). Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies. Int. J. Mol. Sci., 24. 4. Rocha, V.P.C., Quadros, H.C., Fernandes, A.M.S., Gonçalves, L.P., Badaró, R.J.d.S., Soares, M.B.P., and Machado, B.A.S. (2023). An Overview of the Conventional and Novel Methods Employed for SARS-CoV-2 Neutralizing Antibody Measurement. Viruses, 15. 5. Sobhani, K., Cheng, S., Binder, R.A., Mantis, N.J., Crawford, J.M., Okoye, N., Braun, J.G., Joung, S., Wang, M., and Lozanski, G. (2023). Clinical Utility of SARS-CoV-2 Serological Testing and Defining a Correlate of Protection. Vaccines, 11.
|
|