Flow cytometric assay for the detection of anti-SARS-CoV-2 Spike protein antibodies in serum of vaccinated volunteers
Introduction. The determination of antibodies against the Spike (S) protein of the novel coronavirus is widely used to confirm current or past infection with SARS-CoV-2, and as an indicator of the effectiveness of vaccination against COVID-19. The most common method for detecting anti-S-antibodies is enzyme-linked immunosorbent assay (ELISA), which uses a recombinant S-protein. Immunofluorescence followed by flow cytometry provides an alternative approach to detect anti-S-antibodies, where a protein in the native transmembrane conformation is used as the S-antigen. The aim of the study was to develop a method for determining anti-S-antibodies using flow cytometry, and to select the most appropriate method for processing experimental data. Material and methods. The study involved 22 volunteers (7 men and 15 women aged 25 to 70 years, median 48). All volunteers were vaccinated with two doses of the «Sputnik V» vaccine between January and February 2021. Donor sera samples were collected before vaccination with «Sputnik V» and 3 months after vaccination. 5 volunteers had already had a mild form of COVID-19 before the time of vaccination. The remaining 17 volunteers did not encounter the SARS-CoV-2. Antibodies against S-protein were determined by immunofluorescence with registration on a flow cytometer. HEK293 cells were transiently transfected with a plasmid encoding the wild type S-protein which was used as target. Transfection was performed by the calcium phosphate method. Cells were incubated with serially diluted sera and then stained with anti-IgG-PE and anti-IgM-FITC secondary antibodies. The fluorescence level was measured using a flow cytometer. As a measurement result, the mean fluorescence intensity (MFI) obtained at 1:18 serum dilution, or the area under the titration curve (area under curve, AUC) was used. Anti-RBD-antibodies were determined using enzyme immunoassay, and virus-neutralizing activity using pseudotyped or surrogate virus-neutralization analysis (pVNA and sVNA). Results. Using the developed method, the formation of anti-S antibodies of the IgG and IgM isotypes was shown 3 months after immunization with the «Sputnik V» vaccine. In a simplified version of the method, the relative concentration of antibodies was determined at a single dilution of the test serum by measuring the mean fluorescence intensity (MFI) of the target cells. More reliable results were obtained by construction the titration curve and calculating the area under the curve (AUC). The results thus obtained correlated well with the detection of anti-RBD antibodies by ELISA, as well as with virus neutralization data in pseudotyped and surrogate assays. Conclusion. Flow cytometry is a convenient method for the simultaneous determination of anti-S antibodies of IgG and IgM isotypes in human serum. The advantages of the method include the fact that the S-protein is presented in a native transmembrane conformation. After minor modification, the established method can be used to determine the level of anti-S-anti-bodies against mutant variants of SARS-CoV-2.