Limited specificity of commercially available SARS-CoV-2 IgG ELISAs in serum samples of African origin

Abstract

Objectives: Specific serological tests are mandatory for reliable SARS-CoV-2 diagnostics and seroprevalence studies. Here, we assess the specificities of four commercially available SARS-CoV-2 IgG ELISAs in serum/plasma panels originating from Africa, South America, and Europe.

Methods: 882 serum/plasma samples collected from symptom-free donors before the COVID-19 pandemic in three African countries (Ghana, Madagascar, Nigeria), Colombia, and Germany were analysed with three nucleocapsid-based ELISAs (Euroimmun Anti-SARS-CoV-2-NCP IgG, EDI™ Novel Coronavirus COVID-19 IgG, Mikrogen recomWell SARS-CoV-2 IgG), one spike/S1-based ELISA (Euroimmun Anti-SARS-CoV-2 IgG), and in-house common cold CoV ELISAs.

Results: High specificity was confirmed for all SARS-CoV-2 IgG ELISAs for Madagascan (93.4-99.4%), Colombian (97.8-100.0%), and German (95.9-100.0%) samples. In contrast, specificity was much lower for the Ghanaian and Nigerian serum panels (Ghana: NCP-based assays 77.7-89.7%, spike/S1-based assay 94.3%; Nigeria: NCP-based assays 39.3-82.7%, spike/S1-based assay 90.7%). 15 of 600 African sera were concordantly classified as positive in both the NCP-based and the spike/S1-based Euroimmun ELISA, but did not inhibit spike/ACE2 binding in a surrogate virus neutralisation test. IgG antibodies elicited by previous infections with common cold CoVs were found in all sample panels, including those from Madagascar, Colombia, and Germany and thus do not inevitably hamper assay specificity. Nevertheless, high levels of IgG antibodies interacting with OC43 NCP were found in all 15 SARS-CoV-2 NCP/spike/S1 ELISA positive sera.

Conclusions: Depending on the chosen antigen and assay protocol, SARS-CoV-2 IgG ELISA specificity may be significantly reduced in certain populations probably due to interference of immune responses to endemic pathogens like other viruses or parasites.

Keywords: Africa; Enzyme-Linked Immunosorbent Assay; SARS-CoV-2; immunoglobulin G; seroepidemiologic studies; specificity.

Figure 1

SARS‐CoV‐2 IgG ELISA results. (a–d) Index values obtained for serum/plasma samples collected before 2019 in three different African countries (Ghana panel 1 (n = 150), Ghana panel 2 (n = 133), Madagascar (n = 167), Nigeria (n = 150)), Colombia (n = 134), and Germany (n = 148) with the Euroimmun (EI) Anti‐SARS‐CoV‐2‐NCP IgG ELISA (a), the Euroimmun (EI) Anti‐SARS‐CoV‐2 IgG ELISA (b), the EDI Novel Coronavirus COVID‐19 IgG ELISA (c), and the Mikrogen recomWell SARS‐CoV‐2 IgG ELISA (d). Diamonds: index values obtained for two IgG positive COVID‐19 patient sera sampled on day 19 post onset of symptoms (dark/light grey: SARS‐CoV‐2 IgG IIFT titre 1:640/1:160) and one negative control serum; error bars: standard deviation of 13 (a, c) and 14 (b, d) independent measurements. Dotted lines: negative and positive cut‐off values; grey shading indicates index values rated as ‘borderline’ according to the manufacturers’ instructions.

Figure 2

Correlation of SARS‐CoV‐2 IgG ELISA test results (NCP‐based tests), exemplified for the Nigerian sample panel (n = 150). (a) Euroimmun (EI) Anti‐SARS‐CoV‐2‐NCP IgG ELISA vs. EDI Novel Coronavirus COVID‐19 IgG ELISA, (b) Euroimmun (EI) Anti‐SARS‐CoV‐2‐NCP IgG ELISA vs. Mikrogen recomWell SARS‐CoV‐2 IgG ELISA, (c) Mikrogen recomWell SARS‐CoV‐2 IgG ELISA vs. EDI Novel Coronavirus COVID‐19 IgG ELISA. Dotted lines: negative and positive cut‐off values; grey shading indicates index values rated as ‘borderline’ according to the manufacturers’ instructions.

Figure 3

(a–c) Correlation of Euroimmun Anti‐SARS‐CoV‐2‐NCP IgG ELISA and Euroimmun Anti‐SARS‐CoV‐2 IgG ELISA test results (NCP‐based vs. spike/S1‐based test). Shown are index values for sample panels Ghana 1 (a), Ghana 2 (b), and Nigeria (c). Triangles indicate spike/S1/NCP IgG positive samples containing IgG antibodies reacting positive with SARS‐CoV‐2 spike/S2 in the Euroimmun line blot. (D) ELISA, sVNT and line blot results for sera testing positive in both the Euroimmun NCP‐ and spike/S1‐based IgG ELISA. Serum samples (Ghana 1 (G1): n = 4, Ghana 2 (G2): n = 3, Nigeria (N): n = 8)) were tested using the SARS‐CoV‐2 sVNT (Genscript) and the Euroline Anti‐SARS‐CoV‐2 Profile IgG (Euroimmun) according to the manufacturer’s instructions. Rating of index values (iv): Euroimmun ELISA: negative: iv < 0.8, borderline; 0.8 ≤ iv < 1.1, positive: iv ≥ 1.1; Line blot: negative: iv < 0.6, borderline: 0.6 ≤ iv < 1.0, positive: iv ≥ 1.0; in‐house ELISA: negative: iv < 0.7, borderline: 0.7 ≤ iv < 1.3, positive: iv ≥ 1.3; sVNT: negative: % inhibition < 20.0, borderline; 20.0 ≤ % inh < 30.0, positive: % inh ≥ 30.0; CTD: C‐terminal domain; dark grey fields: positive; light grey fields: borderline.

Figure 4

Common cold CoVs ELISA results. Index values obtained for the sample panels from Ghana (1: n = 150, 2: n = 133), Madagascar (n = 167), Nigeria (n = 150), Colombia (n = 134), and Germany (n = 148) using an in‐house IgG ELISA protocol employing the C‐terminal dimerisation domain of (a) OC43 NCP, (b) HKU1 NCP, (c) NL63 NCP, and (d) 229E NCP as antigen. Bold numbers: % of samples for which an iv ≥ 1.3 was obtained, numbers in brackets: 95% confidence interval. Grey shading indicates ivs rated as ‘borderline’ (0.7 ≤ iv < 1.3).