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

Abstract

Background: The COVID-19 pandemic necessitates 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 receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before 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 severities of COVID-19 were tested for SARS-CoV-2-specific IgG concentrations.

Results: Our assay discriminated SARS-CoV-2-induced antibodies and those induced by other viruses. The assay specificity was 95.1%-99.0% with sensitivity 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 nonhospitalized 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. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen-specific IgG determination.

Keywords: COVID-19; IgG; RBD; endemic coronavirus; influenza-like Illness (ILI); multiplex bead-based immune assay; nucleoprotein; sensitivity; specificity; spike S1.

Figure 1.

Assay development and validation A, Different lots of antigen-bead conjugations were used to determine the consistency of the mean fluorescence signal of a titrated pooled reference serum. Two different references consisting of pooled sera of COVID-19 patients (Ref 1 and Ref 2) were tested in independent runs designated A, B or C. B, The reference and serum samples were serially diluted to test parallelism for reliable quantification of antibody concentrations. C, Samples were tested in 2 independent runs by different technicians using different bead and reference batches to test robustness of the triplex assay. Concentrations in AU/mL are shown. Abbreviations: AU, arbitrary units; MFI, mean fluorescence intensity; N, nucleoprotein; RBD, receptor binding domain; S1, spike protein subunit 1.

Figure 2.

Ability of the assay to identify COVID-19 patients. A, Control sera (n = 408) and COVID-19 sera (n = 115) collected after day 10 of symptoms were tested and compared for concentrations of IgG. Median concentration and 95% confidence intervals are shown. B, The sera tested in (A) were analyzed by ROC. C, The ROC data were used to determine Youden J statistic cutoff (lower cutoff) and a specificity-optimized cutoff of at least 98.5% specificity (higher cutoff). Abbreviations: AU, arbitrary unit; IgG, immunoglobulin G; N, nucleoprotein; RBD, receptor binding domain; ROC, receiver operator characteristic; S1, spike protein subunit 1.

Figure 3.

Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A, Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B, Laboratory-confirmed viral infections (see Supplementary Table 2) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.

Figure 4.

Kinetics of antibody production after disease onset in hospitalized and nonhospitalized COVID-19 patients. Paired samples were analyzed to identify changes in IgG concentrations in hospitalized (A) and nonhospitalized (B) COVID-19 patients. C, The log-transformed concentration data of the samples shown in (A) and (B) were fitted with a 4-parameter nonlinear least squared fit. D, Of each patient with paired samples available, 1 sample was selected randomly and data were fitted to estimate the slope, R² of the fits, and the difference between the fitted lines determined. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; IgG, immunoglobulin G; N, nucleoprotein; RBD, receptor binding domain; S1, spike protein subunit 1.