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

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  • Kyosei Yuta
    Department of Biology, Waseda University
  • Namba Mayuri
    Department of Biology, Waseda University
  • Yamura Sou
    Department of Biology, Waseda University
  • Watabe Satoshi
    Waseda Research Institute for Science and Engineering, Waseda University
  • Yoshimura Teruki
    School of Pharmaceutical Sciences, Health Sciences University of Hokkaido
  • Sasaki Tadahiro
    Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University
  • Shioda Tatsuo
    Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University
  • Ito Etsuro
    Department of Biology, Waseda University Waseda Research Institute for Science and Engineering, Waseda University Graduate Institute of Medicine, Kaohsiung Medical University

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<p>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.</p>

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