A highly sensitive and specific SARS-CoV-2 spike- and nucleoprotein-based fluorescent multiplex immunoassay (FMIA) to measure IgG, IgA and IgM class antibodies (preprint)

ABSTRACT

Background. Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. Methods. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organisation (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralisation test (MNT). We also compared the MNT results of two laboratories. Results. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13-150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA 100% specificity and sensitivity were obtained for a shorter time window (13-36 and 13-28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralising antibodies (NAbs). Anti-spike IgG concentrations correlated strongly ({rho}=0.77-0.84, P<2.2x10-16) with NAb titers. The NAb titers of the two laboratories displayed a very strong correlation ({rho}=0.95, P<2.2x10-16). Discussion. Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against WHO international standard did not, however, improve the comparability of FMIA and EIA results. keywords SARS-CoV-2, COVID-19, antibody, immunoassay, nucleoprotein, spike glycoprotein, WHO international standard, neutralising antibodies, microneutralisation, receptor-binding domain