A fluorescent microspheres-based microfluidic test system for the detection of immunoglobulin G to SARS-CoV-2

Abstract

Background: The pandemic of the new coronavirus infection, COVID-19, is currently ongoing in the world. Over the years, the pathogen, SARS-CoV-2, has undergone a series of mutational genome changes, which has led to the spread of various genetic variants of the virus. Meanwhile, the methods used to diagnose SARS-CoV-2, to establish the disease stage and to assess the immunity, are nonspecific to SARS-CoV-2 variants and time-consumable. Thus, the development of new methods for diagnosing COVID-19, as well as their implementation in practice, is currently an important direction. In particular, application of systems based on chemically modified fluorescent microspheres (with a multiplex assay for target protein molecules) opens great opportunities.

Aim: development of a microfluidic diagnostic test system based on fluorescent microspheres for the specific detection of immunoglobulins G (IgG) to SARS-CoV-2.

Methods: A collection of human serum samples was characterized using enzyme-linked immunosorbent assay (ELISA) and commercially available reagent kits. IgG to SARS-CoV-2 in the human serum were detected by the developed immunofluorescent method using microspheres containing the chemically immobilized RBD fragment of the SARS-CoV-2 (“Kappa” variant) viral S-protein.

Results: The level of IgG in the blood serum of recovered volunteers was 9-300 times higher than that in apparently healthy volunteers, according to ELISA (p<0.001). Conjugates of fluorescent microspheres with the RBD-fragment of the S-protein, capable of specifically binding IgG from the blood serum, have been obtained. The immune complexes formation was confirmed by the fluorescence microscopy data; the fluorescence intensity of secondary antibodies in the immune complexes formed on the surface of microspheres was proportional to the content of IgG (r 0.963). The test system had a good predictive value (AUC 70.3%).

Conclusion: A test system has been developed, based on fluorescent microspheres containing the immobilized RBD fragment of the SARS-CoV-2 S-protein, for the immunofluorescent detection of IgG in the human blood serum. When testing the system on samples with different levels of IgG to SARS-CoV-2, its prognostic value was shown. The obtained results allow us to present the test system as a method to assess the level of immunoglobulins to SARS-CoV-2 in the human blood serum for the implementation in clinical practice. The test system can also be integrated into various microfluidic systems to create chips and devices for the point-of-care diagnostics.

Keywords

enzyme-linked immunosorbent assay / ELISA / COVID-19 testing / personalized medicine

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Funding

Научное исследование проведено при поддержке гранта Президента Российской ФедерацииThis work was supported by the grant of the President of Russian Federation(МК-2988.2022.3)

RIGHTS & PERMISSIONS

Shakurov R.I., Shansky Y.D., Prusakov K.A., Sizova S.V., Dudik S.P., Plotnikova L.V., Manuvera V.A., Klinov D.V., Lazarev V.N., Bespyatykh J.A., Basmanov D.V.