Fcγ-Receptor-Based Enzyme-Linked Immunosorbent Assays for Sensitive, Specific, and Persistent Detection of Anti-SARS-CoV-2 Nucleocapsid Protein IgG Antibodies in Human Sera

Abstract

Sensitive and specific serological tests are mandatory for epidemiological studies evaluating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence as well as coronavirus disease 2019 (COVID-19) morbidity and mortality rates. The accuracy of results is challenged by antibody waning after convalescence and by cross-reactivity induced by previous infections with other pathogens. By employing a patented platform technology based on capturing antigen-antibody complexes with a solid-phase-bound Fcγ receptor (FcγR) and truncated nucleocapsid protein as the antigen, two SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISAs), featuring different serum and antigen dilutions, were developed. Validation was performed using a serum panel comprising 213 longitudinal samples from 35 COVID-19 patients and a negative-control panel consisting of 790 pre-COVID-19 samples from different regions of the world. While both assays show similar diagnostic sensitivities in the early convalescent phase, ELISA 2 (featuring a higher serum concentration) enables SARS-CoV-2 IgG antibody detection for a significantly longer time postinfection (≥15 months). Correspondingly, analytical sensitivity referenced to indirect immunofluorescence testing (IIFT) is significantly higher for ELISA 2 in samples with a titer of ≤1:640; for high-titer samples, a prozone effect is observed for ELISA 2. The specificities of both ELISAs were excellent not only for pre-COVID-19 serum samples from Europe, Asia, and South America but also for several challenging African sample panels. The SARS-CoV-2 IgG FcγR ELISAs, methodically combining antigen-antibody binding in solution and isotype-specific detection of immune complexes, are valuable tools for seroprevalence studies requiring the (long-term) detection of anti-SARS-CoV-2 IgG antibodies in populations with a challenging immunological background and/or in which spike-protein-based vaccine programs have been rolled out.

Keywords: immunoassay; immunoglobulins; infectious disease; laboratory methods and tools; viral diseases.

FIG 1

Prokaryotic expression and purification of N-terminally truncated SARS-CoV-2 NCP. (A) Schematic representation of the fusion protein His₆–GST–3C–SARS-CoV-2 NCPΔ246 (calculated molecular weight, 47.3 kDa). (B) Total lysates preinduction (pre) and postinduction (post) and soluble (supernatant [SN]) and insoluble (pellet [P]) lysate fractions. (C) Eluate (E) and matrix (M) after on-column cleavage. (D) Amino acid sequence comparison of truncated CoV NCPs. Above the diagonal/light gray shading are identity scores (percent), and below the diagonal/dark gray shading are similarity scores (percent). MERS, Middle East respiratory syndrome.

FIG 2

Detection range. WHO international standard plasma 20/136 (1,000 BAU/mL) was serially diluted with serum dilution buffer (SDB) (1× PBS [pH 7.4], 0.05% ProClin 300, 0.01% phenol red) to simulate samples with antibody concentrations ranging between 0.1 BAU/mL and 1,000 BAU/mL. Simulated samples were tested with ELISA 1 (open circles) (final in-well dilutions of 1:100 for samples and 1:20,000 for the conjugate) and ELISA 2 (filled circles) (final in-well dilutions of 1:2 for samples and 1:50,000 for the conjugate). Dotted lines indicate A₄₅₀-A₆₂₀ values of 0.2 and 0.4, respectively. Solid lines indicate the linear regression fit.

FIG 3

Diagnostic sensitivity. Longitudinal serum samples from 35 COVID-19 patients were analyzed using ELISA 1 (A and C) and ELISA 2 (B and D). (A and B) Trajectories (individual patients; 1 to 13 samples/patient; n = 213). (C and D) Stratification according to months after the onset of symptoms (≤1 sample/patient/category; n = 146). Dotted lines indicate A₄₅₀-A₆₂₀ values of 0.2 and 0.4. HD, healthy donors (Germany) (n = 139). Filled dots indicate postvaccination samples (spike-based vaccine).

FIG 4

Analytical sensitivity. (A and B) Longitudinal serum samples from 35 COVID-19 patients were analyzed by IgG IIFT. Filled dots indicate postvaccination samples (spike-based vaccine). (A) Trajectories (individual patients; 1 to 13 samples/patient; n = 213); (B) stratification according to months after the onset of symptoms (≤1 sample/patient/category; n = 146). (C and D) Results of ELISA 1 (C) and ELISA 2 (D) for prevaccination samples stratified according to IIFT titers (≤1 sample/patient/category; n = 109). Dotted lines indicate A₄₅₀-A₆₂₀ values of 0.2 and 0.4.