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However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg\/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59\/60) sensitivity and 100% (25\/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries.<\/jats:p>","DOI":"10.3390\/v15091894","type":"journal-article","created":{"date-parts":[[2023,9,8]],"date-time":"2023-09-08T07:52:11Z","timestamp":1694159531000},"page":"1894","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling"],"prefix":"10.3390","volume":"15","author":[{"given":"Po-Kai","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Biology, Waseda University, Tokyo 162-8480, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jyun-Hao","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Biology, Waseda University, Tokyo 162-8480, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2547-0987","authenticated-orcid":false,"given":"Liang-Yin","family":"Ke","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 80708, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2657-9298","authenticated-orcid":false,"given":"Jun-Kai","family":"Kao","sequence":"additional","affiliation":[{"name":"Frontier Molecular Medical Research Center in Children, Changhua Christian Children\u2019s Hospital, Changhua 50006, Taiwan"},{"name":"Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung 402204, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chang-Hua","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung 402204, Taiwan"},{"name":"Changhua Christian Hospital, Changhua 50006, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rei-Cheng","family":"Yang","sequence":"additional","affiliation":[{"name":"Frontier Molecular Medical Research Center in Children, Changhua Christian Children\u2019s Hospital, Changhua 50006, Taiwan"},{"name":"Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung 80756, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Teruki","family":"Yoshimura","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Health Science University of Hokkaido, Hokkaido 061-0293, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1877-6566","authenticated-orcid":false,"given":"Etsuro","family":"Ito","sequence":"additional","affiliation":[{"name":"Department of Biology, Waseda University, Tokyo 162-8480, Japan"},{"name":"Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2226-8916","authenticated-orcid":false,"given":"Jih-Jin","family":"Tsai","sequence":"additional","affiliation":[{"name":"Tropical Medicine Center, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan"},{"name":"School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan"},{"name":"Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1128\/CMR.11.3.480","article-title":"Dengue and dengue hemorrhagic fever","volume":"11","author":"Gubler","year":"1998","journal-title":"Clin. 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