key: cord-0834068-grs1zpag authors: Schöler, Lara; Le-Trilling, Vu Thuy Khanh; Eilbrecht, Mareike; Mennerich, Denise; Anastasiou, Olympia E.; Krawczyk, Adalbert; Herrmann, Anke; Dittmer, Ulf; Trilling, Mirko title: A novel in-cell ELISA assay allows rapid and automated quantification of SARS-CoV-2 to analyse neutralizing antibodies and antiviral compounds date: 2020-06-05 journal: bioRxiv DOI: 10.1101/2020.06.05.135806 sha: ac00dfd3b6aa31b6ce5c52b87fa8caf751a1f778 doc_id: 834068 cord_uid: grs1zpag 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. By the time of writing, more than 6.3 million people experienced a laboratory confirmed infection 52 with the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and more than 378,000 53 people died while having coronavirus infectious disease 19 . First surveillance studies 54 and calculations of excess mortality rates indicate that the precise number of infections and the 55 true number of fatalities exceed above-mentioned numbers by far. Most SARS-CoV-2 infections lead to mild or moderate illnesses. However, a considerable fraction 71 of cases proceeds to severe pneumonia or life-threatening acute respiratory distress syndrome. Elderly individuals and people with pre-existing comorbidities such as impaired immunity, chronic 73 respiratory diseases, cardiovascular diseases, and cancer are more prone to suffer from severe 74 COVID-19. The case fatality rate (CFR) is difficult to calculate in the midst of the pandemic. Depending on the age, CFR estimates of up to 18.4% for individuals older than 80 years and 1.38% 76 (range 1.23 -1.53) for the general population have been reported (6). Given the extent, pace, and severity of the COVID-19 pandemic, diagnostics departments even in 78 countries with highly developed medical systems struggle to provide sufficient and timely test 79 capacities. Since nucleic acid-based pathogen detection, usually by quantitative RT-PCR based on 80 naso-or oropharyngeal swabs, has a very short window of opportunity, assays detecting long-81 lasting immune responses such as antibodies are required to monitor virus spread and to estimate 82 potential herd immunity. With some delay, most infected individuals raise a detectable humoral immune response including 84 specific immunoglobulins (Ig) M, IgA, and IgG (7-9). Neutralizing antibodies (NAbs) bind and 85 abrogate the function of viral proteins such as the SARS-CoV-2 Spike (S) protein that are essential 86 for virus entry into host cells, e.g., through recognition of the cognate receptor ACE2 (10). Accordingly, monoclonal NAbs exhibit strong therapeutic and prophylactic efficacies in SARS- CoV-2-infected human ACE2-transgenic mice (11). A recent vaccination study conducted in non-89 human primates identified NAbs as correlate of protection (12), indicating that a human SARS- to-noise ratio favourable for icELISA (Fig. 1A) . A comparison of Vero E6 and human Caco-2 143 cells revealed that the icELISA is applicable to different cells (Fig. 1B) . ELISAs have been compared to PRNTs (e.g., (28)). To validate the novel icNT, 20 sera -10 186 positive for SARS-CoV-2-specific IgG as determined using the Euroimmun ELISA (ELISA ratio: 187 1.26 to 11.39) and 10 ELISA-negative sera (ELISA ratio < 0.9) -were compared side-by-side by 188 icNT and standard PRNT using 200 PFU/well (Fig. 3A) . 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