Flow cytometry detection of sustained humoral immune response (IgG + IgA) against native spike glycoprotein in asymptomatic/mild SARS-CoV-2 infection

Abstract

SARS-CoV-2 is the virus that causes the disease called COVID-19, which has caused the worst pandemic of the century. Both, to know the immunological status of general population and to evaluate the efficacy of the vaccination process that is taking place around the world, serological tests represent a key tool. Classic serological tests, based on colorimetric techniques, such as ELISA or CLIA, continue to be the most widely used option. However, a real improvement in results is still needed. We developed a highly sensitive and specific FCM assay that allows the detection of IgG and IgA antibodies, directed against the native and functional S-protein of SARS-CoV-2 exposed on the membrane of a transfected cell line, up to 8 months after infection.

Performance of a flow cytometry-based immunoassay for detection of antibodies binding to SARS-CoV-2 spike protein

“…In this study we compared the performance of an in-house-developed quantitative FCI 12 , 13 with the SARS-CoV-2 trimericS-IgG CLIA from Diasorin and Roche RBD-specific IgG/IgM antibody ECLIA for serological diagnosis of SARS-CoV-2 infection in patients with either acute or convalescent COVID-19. The latter two have been reported to measure serum/plasma antibody levels that correlate with those quantified by virus neutralization assays, using either wild type SARS-CoV-2 or lentiviral-S-pseudotyped virions 8 – 11 .…”

“…We measured IgG and IgA antibody levels by FCI as previously described in detail 12 , 13 . Previously published data 12 found detection of SARS-CoV-2-S-binding IgM to be less consistent and reliable.…”

“…Samples were then washed and acquired on an Omnicyt flow cytometer (Cytognos S.L, Salamanca, Spain) and analyzed using the Infinicyt 2.0 software (Cytognos SL). Flow cytometer MFI target values were established in the 5th peak of Rainbow beads (Cytognos SL), according to manufacturer’s instructions 13 . Particle data was acquired in each instrument run.…”

“…Particle data was acquired in each instrument run. The gating strategy has been previously detailed 13 . A minimum of 50,000 viable events, discarding doublets and debris, were considered for the analyses.…”

“…A new generation of recently launched commercially-available SARS-CoV-2 immunoassays detect antibodies binding to the SARS-CoV-2 S protein in its native (trimeric) conformation or RBD and offer quantitative estimates of antibody levels 8 – 11 , and preliminary results show increased sensitivity for detection of SARS-CoV-2 antibodies, as well as reliable estimates of serum neutralizing activity against SARS-CoV-2. In this context, a flow cytometry-based immunoassay (FCI) has been developed employing Jurkat T cells stably expressing the full-length native S protein, which is reported to be highly specific and display greater sensitivity than various comparative immunoassays targeting recombinant RBD or S subunit proteins 12 , 13 . Here, we evaluated the performance of this FCI against two new-generation immunoassays: Elecsys Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG assay (Diasorin S.p.a, Saluggia, IT), using sera from in- or outpatients with SARS-CoV-2 infection documented by RT-PCR.…”