Desenvolvimento de teste sorológico para COVID-19 por citometria de fluxo

Abstract

The first coronavirus capable of infecting humans was isolated in the 1960s, and for a long time was considered to cause a mild flu condition. Coronaviruses are a family of enveloped viruses, with a single-stranded, non-segmented, positive-sense RNA genome. The envelope is composed of a lipid bilayer to which the spike (S), membrane (M) and envelope (E) proteins are attached. The protein (N) is associated with genomic RNA, and together they make up the nucleocapsid. The flow cytometry technique is capable of identifying several parameters in a single cell or in biological samples. In this work, we started the development of a Cytometric Bead Array (CBA) assay for the simultaneous detection of IgM and IgG antibodies specific for the N proteins of SARS-CoV-2 in human samples, either for diagnosis or for monitoring vaccine efficacy, this method can be easily adapted for the detection of antibodies specific for the spike Protein S1 Receptor-Binding Domain (S1-RBD) in the future. Functional beads CBA A5, kit for performing the CBA flex assay, buffer kit for conjugation of functional beads, mouse monoclonal antibody anti-human IgG conjugated to PE, as well as other materials and reagents necessary for the conjugation of the beads, including Bio-Spin P-30 gel columns and DTT, Sulfo-SMCC, NEM and DMSO reagents. For initial evaluation of the sensitivity and specificity of the test, we used serum samples from rabbits immunized with proteins N (positive controls) and S1-RBD (negative controls), as well as non-immunized (negative control). The results demonstrated that the positive and negative samples were properly recognized in the assay, and that there is no non-specific reaction. We used 37 random serum samples obtained from voluntary donors in order to determine whether it would be possible to detect anti-N antibodies in these samples using the CBA assay. As expected, anti-N antibody titers in the population are low, The only vaccine that so far can produce antibodies against N protein is CoronaVac, from Sinovac, and even so, according to a study by Bochnia 16 (2021), which is reflected in low fluorescence intensity compared to the positive control with commercial antibody. Considering everything that was observed in this work, we can conclude that flow cytometry is an excellent diagnostic method for serological follow-up of the population, either for vaccination follow-up or after infection.Due to the promising results, the test will continue to be developed at the Immunochemistry Laboratory.