Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2

Abstract

Serological tests developed for COVID-19 diagnostic are based on antibodies specific for SARS-CoV-2 antigens. Most of the antigens consist of a fragment or a whole amino acid sequence of the nucleocapsid or spike proteins. We evaluated a chimeric recombinant protein as an antigen in an ELISA test, using the most conserved and hydrophilic portions of the S1-subunit of the S and Nucleocapsid (N) proteins. These proteins, individually, indicated a suitable sensitivity of 93.6 and 100% and a specificity of 94.5 and 91.3%, respectively. However, our study with the chimera containing S1 and N proteins of SARS-CoV-2 suggested that the recombinant protein could better balance both the sensitivity (95.7%) and the specificity (95.5%) of the serological assay when comparing with the ELISA test using the antigens N and S1, individually. Accordingly, the chimera showed a high area under the ROC curve of 0.98 (CI 95% 0.958-1). Thus, our chimeric approach could be used to assess the natural exposure against SARS-CoV-2 virus over time, however, other tests will be necessary to better understand the behaviour of the chimera in samples from people with different vaccination doses and/or infected with different variants of the virus.

Keywords: COVID-19; Chimeric protein; Diagnostic; ELISA test; SARS-CoV-2.

Fig. 1

ROC analysis of ELISA tests with each antigen: A- N protein, B- S1 protein, C- S protein, D- S-RBD protein, E- S2 protein. Area Under the ROC Curve (AUC), confidence interval (CI).

Fig. 2

ROC analysis using S1/N chimeric protein antigen for the ELISA test using the serological rapid test as reference. Area under ROC curve (AUC), confidence interval (CI).

Fig. 3

ROC analysis with Anti-SARS-CoV-2 (IgG) ELISA (Euroimmun). Area under ROC curve (AUC), confidence interval (CI).