SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

Abstract

Background The COVID-19 pandemic necessitates a better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population. Methods Spike protein subunits S1 and RBD, and Nucleoprotein were coupled to distinct microspheres. Sera collected before the emergence of SARS-CoV-2 (N=224), and of non-SARS-CoV-2 influenza-like illness (N=184), and laboratory-confirmed cases of SARS-CoV-2 infection (N=115) with various severity of COVID-19 were tested for SARS-CoV-2-specific concentrations of IgG. Results Our assay discriminated SARS-CoV-2-induced antibodies and those induced by other viruses. The assay obtained a specificity between 95.1 and 99.0% with a sensitivity ranging from 83.6-95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to non-hospitalized cases. Conclusions The bead-based serological assay for quantitation of SARS-CoV-2-specific antibodies proved to be robust and can be conducted in many laboratories. Finally, we demonstrated that testing of antibodies against different antigens increases sensitivity and specificity compared to single antigen-specific IgG determination.

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

“…Ng and colleagues identified such cross-reacting antibodies against non-RBD S-protein epitopes in sera originating from the pre-pandemic era, which also demonstrated activity in the neutralization assay [44]. Cross-reacting antibodies due to endemic common cold coronavirus disease have also been demonstrated by others [45][46][47][48][49]. It remains to be seen whether an immune-reactivity to non-S1-RBD spike protein lowers the risk of contracting COVID-19 during the pandemic.…”

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

“…The aim of this study was to assess the clinical performance of a multiplex assay designed to detect S1-, S2-, N-, RBD- and NTD-directed antibodies. The major promises of multiplex methods for anti-SARS-CoV-2 antibodies included increased performance in terms of sensitivity and of specificity [ 25 , 26 , 27 , 28 ], which is in line with the orthogonal testing algorithm proposed by the Centers for Disease Control and Prevention (CDC). The simultaneous detection of several antigens could also permit to predict the disease outcome by identifying various antibody signatures and allow to assess the immunological response to future vaccines with more objectivity [ 29 ].…”

Dual-Antigen System Allows Elimination of False Positive Results in COVID-19 Serological Testing

“…Testing for antibodies specific to different viral antigens appeared to us a practical approach to increase confidence of the immune response assessment. It was recently shown that a multiple-antigen approach can indeed increase specificity of testing [ 13 ]. The focus of this study was to develop an accurate, robust, and reproducible multi-antigen testing approach which would allow for maximum specificity in the ELISA format, while maintaining an adequate sensitivity level.…”

“…In other approaches involving negative and positive training sample sets, statistical methods, such as the Youden J statistic cutoff, are used to determine absolute antibody threshold concentrations, and in following experiments absolute concentration values of studied samples to said threshold are calculated and compared [ 13 ]. However, this also may not be convenient during large-scale analysis, e.g., population screening, where the robustness of the system is crucial and the qualitative tests may show significant bias when switching between laboratories, equipment, and operators.…”