Clinical performance of different SARS‐CoV‐2 IgG antibody tests

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) serological assays are urgently needed for rapid diagnosis, contact tracing, and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples, and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two enzyme‐linked immunosorbent assay (ELISA) assays (Euroimmun SARS‐CoV‐2 IgG and Vircell COVID‐19 ELISA IgG) in comparison to one lateral flow assay (FaStep COVID‐19 IgG/IgM Rapid Test Device) and two in‐house developed assays (immunofluorescence assay [IFA] and plaque reduction neutralization test [PRNT]). We tested follow up serum/plasma samples of individuals polymerase chain reaction‐diagnosed with COVID‐19. Most of the SARS‐CoV‐2 samples were from individuals with moderate to the severe clinical course, who required an in‐patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5‐9) and from 93.8% to 100% for the later period (days 10‐18).

For all 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. 3 Currently, there are many S protein-based commercially or in-house developed assays available, but there is limited data on how these tests perform with clinical samples, and if the detected IgG antibodies provide protective immunity. This study aims to provide a quick overview on some of these assays (two commercially available enzyme-linked immunosorbent assay [ELISA] assays, one lateral flow assay and two in-

| Enzyme-linked immunosorbent assay
The CE certified versions of the Euroimmun SARS-CoV-2 IgG ELISA (Euroimmun, Lübeck, Germany) and Vircell COVID-19 ELISA IgG (Vircell Spain S.L.U., Granada, Spain) were used, in an identical manner, according to the manufacturer's recommendation. Both ELISA assays use SARS-CoV-2 recombinant antigen from spike glycoprotein (S protein) and the Vircell ELISA additionally Nucleocapsid (N protein). Samples were diluted 1:101 or 1:20, respectively, in sample buffer and incubated at 37°C for 60 minutes in a 96-well microtiter plate followed by each protocol washing and incubation cycles, including controls and required reagents. Optical density was measured for both assays at 450 nm using a Virclia microplate reader (Vircell Spain S.L.U., Granada, Spain). The titers were calculated and results interpreted according to each manufacturer's protocol.

| DISCUSSION
In terms of sensitivity, our data are consistent with previously published data. In a study from Liu et al, 4 using an rS-based ELISA assay, the group found SARS-CoV-2 IgG antibodies in less than 60% of the samples from days 6 to 10 after disease onset. The sensitivity increased to more than 90% in samples from days 16 to 20 4  An important finding of our study is, that (with the exception of sample 1) all detected SARS-CoV-2 IgG antibodies in the analyzed cohort, using the commercially available assays examined, demonstrated neutralizing (potentially protective) properties in the PRNT.
The screening for SARS-CoV-2 IgG antibodies [especially for potential protective IgG antibodies against the S protein 8 using ELISA or lateral flow assays is more convenient and practicable than using the handson-and time-intensive IFA or PRNT, which can only be performed by experienced personnel, and the PRNT, only in a BSL-3 laboratory.
ELISA-based assays can be automated and used for larger sample sizes. Lateral flow assays can be used by less experienced personnel in a point-of-care setting, generating results in a short time. Some samples, however, were only detected with the IFA and PRNT as the gold standard. The titer needed for potential protective immunity is not yet (officially) defined. In one study, it is reported, that an individual cleared SARS-CoV-2 without developing antibodies up to 46 days after illness. 9 The mechanism of immunity, especially of protective immunity (if applicable) and how long it will last, needs to be further investigated. Besides a humoral-mediated immune response, there is evidence that T-cell mediated immunity plays a role. 10 Most of the SARS-CoV-2 samples analyzed in this study were from KOHMER ET AL. At the moment, however, the PRNT is still the method of choice in detecting potential neutralizing antibodies.