ABSTRACT
The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serology ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Using commercially available nucleocapsid protein-specific antibodies, viral infection could easily be quantified in human and highly permissive Vero E6 cells by icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose-dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icNT was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. The SARS-CoV-2 icELISA test allows rapid (<48h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs as well as antiviral drugs, using reagents and equipment present in most routine diagnostics departments. We propose the icELISA and the icNT for COVID-19 research and diagnostics.
