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. 2020 Dec 9;9(12):3989.
doi: 10.3390/jcm9123989.

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study

Anna Schaffner  1 , Lorenz Risch  2   3   4 , Stefanie Aeschbacher  5 , Corina Risch  2 , Myriam C Weber  1 , Sarah L Thiel  1 , Katharina Jüngert  1 , Michael Pichler  1 , Kirsten Grossmann  2   3 , Nadia Wohlwend  2 , Thomas Lung  2 , Dorothea Hillmann  2 , Susanna Bigler  6 , Thomas Bodmer  6 , Mauro Imperiali  7 , Harald Renz  8 , Philipp Kohler  9 , Pietro Vernazza  9 , Christian R Kahlert  9   10 , Raphael Twerenbold  5   11 , Matthias Paprotny  1 , David Conen  12 , Martin Risch  13
Affiliations

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study

Anna Schaffner et al. J Clin Med. .

Abstract

Pan-immunoglobulin assays can simultaneously detect IgG, IgM and IgA directed against the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 S1-RBD Ig). In this work, we aim to evaluate a quantitative SARS-CoV-2 S1-RBD Ig electrochemiluminescence immunoassay (ECLIA) regarding analytical, diagnostic, operational and clinical characteristics. Our work takes the form of a population-based study in the principality of Liechtenstein, including 125 cases with clinically well-described and laboratory confirmed SARS-CoV-2 infection and 1159 individuals without evidence of coronavirus disease 2019 (COVID-19). SARS-CoV-2 cases were tested for antibodies in sera taken with a median of 48 days (interquartile range, IQR, 43-52) and 139 days (IQR, 129-144) after symptom onset. Sera were also tested with other assays targeting antibodies against non-RBD-S1 and -S1/S2 epitopes. Sensitivity was 97.6% (95% confidence interval, CI, 93.2-99.1), whereas specificity was 99.8% (95% CI, 99.4-99.9). Antibody levels linearly decreased from hospitalized patients to symptomatic outpatients and SARS-CoV-2 infection without symptoms (p < 0.001). Among cases with SARS-CoV-2 infection, smokers had lower antibody levels than non-smokers (p = 0.04), and patients with fever had higher antibody levels than patients without fever (p = 0.001). Pan-SARS-CoV-2 S1-RBD Ig in SARS-CoV-2 infection cases significantly increased from first to second follow-up (p < 0.001). A substantial proportion of individuals without evidence of past SARS-CoV-2 infection displayed non-S1-RBD antibody reactivities (248/1159, i.e., 21.4%, 95% CI, 19.1-23.4). In conclusion, a quantitative SARS-CoV-2 S1-RBD Ig assay offers favorable and sustained assay characteristics allowing the determination of quantitative associations between clinical characteristics (e.g., disease severity, smoking or fever) and antibody levels. The assay could also help to identify individuals with antibodies of non-S1-RBD specificity with potential clinical cross-reactivity to SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; area under the curve; coronavirus; diagnostic accuracy; immunoassay; predictive values; sensitivity; serology; specificity.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Figures

Figure 1
Figure 1
SARS-CoV-2 S1-receptor binding domain (RBD) Ig levels in patients with (n = 125) and without SARS-CoV-2 infection (n = 1159) on a logarithmic scale. Boxplots display medians and interquartile range; whiskers are shown according to Tukey’s.
Figure 2
Figure 2
Pan-SARS-CoV-2 S1-RBD Ig levels upon serial dilution (1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128) of a pool serum with a concentration of 105 U/mL are displayed. The dotted line displays the manufacturer’s cut-off (0.8 U/mL).
Figure 3
Figure 3
Receiver operating characteristic curves of different antibody assays measuring immunoglobulins directed against spike protein. The optimum decision point for SARS-CoV-2 S1-RBD antibodies was a cuttoff index COI > 1.0 U/L.
Figure 4
Figure 4
Pan-SARS-CoV-2 S1-RBD antibody titers in patients with SARS-CoV-2 infection stratified according to the severity of disease (hospitalized patients, n = 12; symptomatic outpatients, n = 104; asymptomatic individuals, n = 9). Medians and interquartile range are shown; whiskers are shown according to Tukey’s. *** p < 0.001.
Figure 5
Figure 5
Course of antibody levels of pan-SARS-CoV-2 S1-RBD Ig in patients with SARS-CoV-2. Patients with ascending levels are shown in blue, patients with descending antibody levels are displayed in green, and patients with unchanged antibody levels are given in orange. In total, 15 patients had unchanged results at the upper limit of quantification at 250 U/mL.
Figure 6
Figure 6
Venn diagram displaying negative anti-spike protein antibody assay results in 125 patients with SARS-CoV-2 infection.
Figure 7
Figure 7
Venn diagram displaying positive anti-spike protein antibody assay results in 1159 individuals without SARS-CoV-2 infection. Manufacturers’ cutoffs were used as a criterion to adjudicate test positivity.
Figure 8
Figure 8
Venn diagram displaying positive anti-spike protein antibody assay results in 1159 individuals without SARS-CoV-2 infection. Half of the manufacturers’ cutoffs was used as a criterion to adjudicate test positivity.
Figure 9
Figure 9
Positive (PPV; panel (a)) and negative (NPV; panel (b)) predictive values of pan-SARS-CoV-2 S1-RBD Ig at two different cut-offs (half and double of the manufacturers’ cutoff) over the whole range of possible pretest probabilities.
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