key: cord-336535-r3a57m57
authors: Kohmer, Niko; Westhaus, Sandra; Rühl, Cornelia; Ciesek, Sandra; Rabenau, Holger F.
title: Brief clinical evaluation of six high-throughput SARS-CoV-2 IgG antibody assays
date: 2020-06-01
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104480
sha: 
doc_id: 336535
cord_uid: r3a57m57

Serological SARS-CoV-2 assays are urgently needed for diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how recently commercially available, high-throughput immunoassays, using different recombinant SARS-CoV-2 antigens, perform with clinical samples. Focusing on IgG and total antibodies, we demonstrate the performance of four automated immunoassays (Abbott Architect™ i2000 (N protein-based)), Roche cobas™ e 411 analyzer (N protein-based, not differentiating between IgA, IgM or IgG antibodies), LIAISON®XL platform (S1 and S2 protein-based), VIRCLIA® automation system (S1 and N protein-based) in comparison two ELISA assays (Euroimmun SARS-CoV-2 IgG (S1 protein-based) and Virotech SARS-CoV-2 IgG ELISA (N protein-based)) and an in-house developed plaque reduction neutralization test (PRNT). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. When calculating the overall sensitivity, in a time frame of 49 days after first PCR-positivity, the PRNT as gold standard, showed the highest sensitivity with 93.3% followed by the dual-target assay for the VIRCLIA® automation system with 89%. The overall sensitivity in the group of N protein-based assays ranged from 66.7 to 77.8% and in the S protein-based-assays from 71.1 to 75.6%. Five follow-up samples of three individuals were only detected in either an S and/or N protein-based assay, indicating an individual different immune response to SARS-CoV-2 and the influence of the used assay in the detection of IgG antibodies. This should be further analysed. The specificity of the examined assays was ≥ 97%. However, because of the low or unknown prevalence of SARS-CoV-2, the examined assays in this study are currently primarily eligible for epidemiological investigations, as they have limited information in individual testing.

. In addition, the SARS-CoV-2 serostatus of asymptomatic individuals or patients with mild clinical course, who present late (a couple of weeks) after infection, is of interest. Ideally, a positive IgG status will offer a potential immunity, but if so, questions on how long it will last, still remain.

Furthermore for therapeutic or prophylactic approaches, convalescent plasma may be used as vaccines and other drugs are under development (3) . For these purposes, sensitive and especially highly specific antibody assays are needed.

The spike (S) protein of SARS-CoV-2 has shown to be highly immunogenic and is the main target for neutralizing antibodies (4) . Currently there are different spike (S) and/or nucleocapsid (N) proteinbased commercially or in-house developed assays available, but there is limited data on how these tests perform with clinical samples.

This study aims to provide a quick overview on some of these assays (two commercially available ELISA assays, four automated immunoassays and a plaque reduction neutralization test (PRNT)) focusing on the detection and neutralization capacity of IgG antibodies in follow up serum or plasma samples of individuals with PCR-diagnosed infections with SARS-CoV-2. When calculating the overall sensitivity we used the total time frame of 49 days after first PCR-positivity and focussed on the different antigens (S-or N-antigen) used as binding antigen(s) in the assays. Typically, the majority of J o u r n a l P r e -p r o o f

We collected follow up serum or plasma samples (in the following simply stated as samples) from individuals with PCR-diagnosed infections with SARS-CoV-2 (n=45) (TABLE S1) (TABLE S2) . The non-SARS-CoV-2 samples were used to assess potential cross reactivity and the risk of potential false positive results.

Samples were tested within one day, in batches, on multiple commercially available (mostly automated) immunoassay platforms (TABLE 1) according to the manufacturers' protocol.

The Euroimmun SARS-CoV-2 IgG ELISA (Euroimmun, Lübeck, Germany) and Virotech SARS-CoV-2 IgG ELISA (Virotech Diagnostics GmbH, Rüsselsheim, Germany; TABLE 1) were used, in an identical manner, according to the manufacturer's recommendation. Samples were diluted 1:101 in sample buffer and incubated at 37° for 60 or 30 mins, respectively, in a 96-well microtiter plate followed by each protocols' washing and incubation cycles, including controls and required reagents. Optical density (OD) was measured for both assays at 450 nm using the microplate reader of a VIRCLIA® automation system (Vircell Spain S.L.U., Granada, Spain). The signal-to-cut-off ratio was calculated and values expressed according to each manufacturer's protocol. For these purposes, there is a demand for (cost-effective) high-throughput assays, which can be automated and used for large sample sizes. The sensitivity of these assays depends on the used assay and moment of testing in the infection phase (low sensitivity a couple of days after infection vs.

higher sensitivity a couple of weeks after infection (5, 6) . The commercially available assays examined in our study, generated consistent results regarding the detection of SARS-CoV-2-IgG antibodies. The sensitivity (without differentiating the timepoint of sampling) varied within the group of assays using the same antigen as target for the antibodies. While the majority of antibodies are typically produced against the N-protein (which therefore might be the most sensitive target protein), antibodies produced against the S-protein are expected to be more specific and potentially neutralizing.

In the group of N protein-based assays the sensitivity varied from 66.7 to 77.8% and in the S proteinbased assays from 71.1 to 75.6%. This might be due to differences within the used recombinant antigen and/or is a system-inherent feature. The dual target (S1 and N protein-based) assay for the Vircell VIRCLIA® automation system and the PRNT demonstrated the highest sensitivity with 89% and 93.3%, respectively. There is a large discrepancy in the determined sensitivities for the assays examined in our study to the sensitivities according to the manufacturers' specifications and the data described in literature. This is not because of the small examined sample size, but because overall sensitivities (not differentiating between the time-points after positive PCR-testing) were given in this study. This was done for a better comparability of the examined assays in terms of demonstrating the differences in the used antigens of the assays on its ability to detect antibodies, independent from the time point of sampling. As gold standard, the PRNT is hands on-and time-intensive and can J o u r n a l P r e -p r o o f only be performed for smaller sample sizes in a BSL-3 laboratory. However, it is capable to detect neutralizing antibodies. In our study, the antibody titers generated with the commercially available assays correlated well with the PRNT titers. The mechanism of immunity, especially of protective immunity (if applicable) and how long it will last, need to be further investigated. A titer needed for potential protective immunity is not yet (officially) defined. Besides humoral mediated immunity, there is evidence that T-cell mediated immunity plays a role (7) . Interestingly, in samples of three individuals with mild clinical course of COVID-19, examined in our study (1, 2, 3 in The automated immunoassays demonstrated a higher overall sensitivity than the ELISA based assays.

Especially the assay using the S and N protein as antigens showed the highest sensitivity within the group of commercially available assays examined in this study (including samples with individual characteristics). The titers generated with these assays correlated well with the PRNT, demonstrating the neutralizing capacity of detected antibodies. Because of the low prevalence of SARS-CoV-2 at the moment, these assays are currently primarily eligible for epidemiological investigations, as they are only of limited informative value in individual testing.

NK and HR designed the study. NK, CR and SW performed experiments. NK, HR and SC analyzed data. NK and HR wrote the manuscript.

All authors discussed the results and commented on the final manuscript. 

SARS-CoV-2 and Coronavirus Disease 2019: What We Know So Far

Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province

Convalescent plasma transfusion for the treatment of COVID-19: Systematic review

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Antibody Detection and Dynamic Characteristics in Patients with COVID-19

Profile of specific antibodies to SARS-CoV-2: The first report

The trinity of COVID-19: immunity, inflammation and intervention

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease

*two equivocal results (HCoV-OC43, negative control cohort) were considered as negative

**including follow up samples of SARS-CoV

three equivocal results (one HCoV-229E sample

*one equivocal result for one SARS-CoV sample was considered negative