Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion

Conzentino, Marcelo S.; Forchhammer, Karl; Souza, Emanuel M.; Pedrosa, Fábio O.; Nogueira, Meri B.; Raboni, Sônia M.; Rego, Fabiane G. M.; Zanette, Dalila L.; Aoki, Mateus N.; Nardin, Jeanine M.; Fornazari, Bruna; Morales, Hugo M. P.; Celedon, Paola A. F.; Lima, Carla V. P.; Mattar, Sibelle B.; Lin, Vanessa H.; Morello, Luis G.; Marchini, Fabricio K.; Reis, Rodrigo A.; Huergo, Luciano F.

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.