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. 2022 Jun 15;60(6):e0007522.
doi: 10.1128/jcm.00075-22. Epub 2022 May 16.

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

Christina Deschermeier  1 , Christa Ehmen  1 , Ronald von Possel  2   3 , Carolin Murawski  1 , Ben Rushton  1 , John Amuasi  4 , Nimako Sarpong  5 , Oumou Maiga-Ascofaré  5   6 , Raphael Rakotozandrindrainy  7 , Danny Asogun  8 , Yemisi Ighodalo  8 , Lisa Oestereich  2   6 , Sophie Duraffour  2   6 , Meike Pahlmann  2   6 , Petra Emmerich  2   3
Affiliations

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

Christina Deschermeier et al. J Clin Microbiol. .

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.

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Conflict of interest statement

The authors declare a conflict of interest. Research Funding: C. Deschermeier, German Federal Ministry of Education and Research (grant no. 01KI20210) and European Regional Development Fund (ERDF) (grant no. BWF/H/52228/2012/13.10.10-1/3.4,6) to institution; P. Emmerich, German Federal Ministry of Education and Research (grant no. 01KI20210) to institution; L. Oestereich: Leibniz Association (grant no. J59/20218) to institution. Patents: C. Deschermeier, EP3207375; P. Emmerich, EP2492689, EP3207375. Other financial or non-financial interests: C. Deschermeier and B. Rushton are cofounders and shareholders of Panadea Diagnostics GmbH. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript. No other Potential Conflicts of Interest have been declared.

Figures

FIG 1
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
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
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
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.
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