Improved Detection Sensitivity Of An Antigen Test For Sars-Cov-2 Nucleocapsid Proteins With Thio-Nad Cycling

Antigen tests for infectious diseases are inexpensive and easy-to-use, but the limit of detection (LOD) is generally higher than that of PCR tests, which are considered the gold standard. In the present study, we combined a sandwich enzyme-linked immunosorbent assay (ELISA) with thionicotinamide-adenine dinucleotide (thio-NAD) cycling to improve the LOD of antigen tests for coronavirus disease 2019 (COVID-19). For recombinant nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the LOD of our ELISA with thio-NAD cycling was 2.95 x 10(-17 )moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 x 10(4) RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 mu L. The minimum detectable value was smaller than that of the latest antigen test using a fluorescent immunoassay for SARS-CoV-2, indicating the validity of our detection system for COVID-19 diagnosis.