Improved Detection Sensitivity of an Antigen Test for SARS-CoV-2 Nucleocapsid Proteins with Thio-NAD Cycling

• Published in: Biological & pharmaceutical bulletin Vol. 44; no. 9; pp. 1332 - 1336
• Main Authors: Kyosei, Yuta, Namba, Mayuri, Yamura, Sou, Watabe, Satoshi, Yoshimura, Teruki, Sasaki, Tadahiro, Shioda, Tatsuo, Ito, Etsuro
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.09.2021; Japan Science and Technology Agency
• Subjects: Adenine; antigen test; Antigens; coronavirus disease 2019; Coronaviruses; COVID-19; Enzyme-linked immunosorbent assay; Infectious diseases; NAD; nucleocapsid protein; Nucleocapsids; Severe acute respiratory syndrome coronavirus 2; thionicotinamide-adenine dinucleotide cycling; ultrasensitivity; Virions
• ISSN: 0918-6158; 1347-5215; 1347-5215
• DOI: 10.1248/bpb.b21-00387

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 × 10−17 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 × 104 RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 µL. 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.