Methods to evaluate the impact of SARS-CoV-2 nucleocapsid mutations on antigen detection by rapid diagnostic tests

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance. Expression, purification and testing of wild-type and mutant recombinant SARS-CoV-2 nucleocapsid antigens by the lateral flow SARS-CoV-2 antigen tests BinaxNOW™ and Panbio™, an ARCHITECT SARS-CoV-2 antigen prototype automated immunoassay, immunoblot and BIAcore™ (surface plasmon resonance) activity assays.