An Original ELISA-Based Multiplex Method for the Simultaneous Detection of 5 SARS-CoV-2 IgG Antibodies Directed against Different Antigens

Abstract

Strategies to detect SARS-CoV-2 are increasingly being developed. Among them, serological methods have been developed. Nevertheless, although these may present an interesting clinical performance, they are often directed against only one antigen. This study aims at evaluating the clinical performance of an innovative multiplex immunoassay (i.e., CoViDiag assay) detecting simultaneously the presence of antibodies directed against N, S1, S2, RBD and NTD antigens. Sensitivity was evaluated in 135 samples obtained from 94 rRT-PCR confirmed coronavirus disease 2019 (COVID-19) patients. Non-SARS-CoV-2 sera (n = 132) collected before the COVID-19 pandemic with potential cross-reactions to the SARS-CoV-2 immunoassay were included in the specificity analysis. The antibody signature was also studied in hospitalized and non-hospitalized patients. The specificity of the CoViDiag assay was excellent for all antibodies (99.2 to 100%) using adapted cut-offs. None of the false positive samples were positive for more than one antibody. The sensitivity obtained from samples collected 14 days since symptom onset varied from 92.0 to 100.0% depending on the antibody considered. Among samples collected more than 14 days after symptom onset, 12.8, 66.3, 3.5, 9.3, 5.8 and 2.3% were positive for 5, 4, 3, 2, 1 or 0 antibodies, respectively. A trend toward higher antibody titers was observed in hospitalized patient in the early days since symptom onset. However, no significant difference was observed compared to non-hospitalized patients after 14 days since symptom onset. The clinical performance of the CoViDiag 5 IgG assay is sufficient to recommend its use for the detection and the characterization of the antibody signature following SARS-CoV-2 infection. The combination of several antigens in the same test improves the overall specificity and sensitivity of the test. Further research is needed to investigate whether this strategy may be of interest to identify severe disease outcome in patients with SARS-CoV-2 infection.

Keywords: COVID-19; SARS-CoV-2; kinetics; multiplex; serology.

Figure 1

The arbitrary units obtained for each sample as a function of each antibody. Cut-offs of the manufacturer (in black; N and S2 > 15 AU and RBD, NTD and S2 > 10 AU) and optimized cut-offs (in colors) are presented on the Y-axis. NA: not applicable.

Figure 2

Evolution of the positivity rate according to the number of days since the onset of symptoms for each antigen (at least one positive antigen and according to the manufacturer algorithm).

Figure 3

Evolution of the average of the arbitrary units obtained for each antigen in the different periods of time since the onset of the symptoms and representation of the distribution of the samples according to the number of positive antigens.

Figure 4

Antibody levels in hospitalized (H) and non-hospitalized (NH) patients. Samples obtained after 14 days since symptom onset were included. Purple: N-directed IgG, Blue: S1-directed IgG, Green: S2-directed IgG, Orange: RBD-directed IgG. Light colors correspond to non-hospitalized patients. Dark colors correspond to hospitalized patients.

Figure 5

Antibody levels in hospitalized (H) and non-hospitalized (NH) patients. Samples obtained before 14 days since symptom onset were included and stratified into the first and second week. Purple: N-directed IgG, Blue: S1-directed IgG, Green: S2-directed IgG, Orange: RBD-directed IgG. Light colors correspond to non-hospitalized patients. Dark colors correspond to hospitalized patients.