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Accurate serological assays are essential for tracking infection history, evaluating disease severity, and guiding vaccination strategies. However, existing assays are limited in their specificity, sensitivity, and cross-reactivity. Using optical modulation biosensing (OMB) technology and non-structural protein 1 (NS1) antigens from DENV-1\u20133, we developed highly sensitive and quantitative serotype-specific anti-DENV NS1 IgG serological assays. The OMB-based assays offered a wide dynamic range (~4-log), low detection limits (~400 ng\/L), fast turnaround (1.5 h), and a simplified workflow. Using samples from endemic (Vietnam) and non-endemic (Israel) regions, we assessed intra-DENV and inter-Flavivirus cross-reactivity. Each assay detected DENV infection with a 100% sensitivity for the corresponding serotype and 64% to 90% for other serotypes. Cross-reactivity with Zika, Japanese encephalitis, and West Nile viruses ranged from 21% to 65%, reflecting NS1 antigen conservation. Our study provides valuable insights into the cross-reactivity of DENV NS1 antigens widely used in research and highlights the potential of OMB-based assays for quantitative and epidemiological studies. Ongoing efforts should aim to minimize cross-reactivity while maintaining sensitivity and explore integration with complementary platforms for improved diagnostic precision.<\/jats:p>","DOI":"10.3390\/bios15070453","type":"journal-article","created":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T09:45:52Z","timestamp":1752572752000},"page":"453","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Sensitivity and Cross-Reactivity Analysis of Serotype-Specific Anti-NS1 Serological Assays for Dengue Virus Using Optical Modulation Biosensing"],"prefix":"10.3390","volume":"15","author":[{"given":"Sophie","family":"Terenteva","sequence":"first","affiliation":[{"name":"Faculty of Engineering, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Max and Anna Webb Street, Ramat Gan 5290002, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5510-3503","authenticated-orcid":false,"given":"Linoy","family":"Golani-Zaidie","sequence":"additional","affiliation":[{"name":"Faculty of Engineering, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Max and Anna Webb Street, Ramat Gan 5290002, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shira","family":"Avivi","sequence":"additional","affiliation":[{"name":"Faculty of Engineering, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Max and Anna Webb Street, Ramat Gan 5290002, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaniv","family":"Lustig","sequence":"additional","affiliation":[{"name":"Central Virology Laboratory, Israel Ministry of Health, Chaim Sheba Medical Centre, Tel-HaShomer, Ramat Gan 5262000, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7675-0591","authenticated-orcid":false,"given":"Victoria","family":"Indenbaum","sequence":"additional","affiliation":[{"name":"Central Virology Laboratory, Israel Ministry of Health, Chaim Sheba Medical Centre, Tel-HaShomer, Ramat Gan 5262000, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravit","family":"Koren","sequence":"additional","affiliation":[{"name":"Central Virology Laboratory, Israel Ministry of Health, Chaim Sheba Medical Centre, Tel-HaShomer, Ramat Gan 5262000, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tran Mai","family":"Hoa","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Parasitology, Faculty of Medical Technology, Hanoi Medical University, Hanoi 115600, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tong Thi Kim","family":"Tuyen","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Parasitology, Faculty of Medical Technology, Hanoi Medical University, Hanoi 115600, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ma Thi","family":"Huyen","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Parasitology, Faculty of Medical Technology, Hanoi Medical University, Hanoi 115600, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nguyen Minh","family":"Hoan","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Parasitology, Faculty of Medical Technology, Hanoi Medical University, Hanoi 115600, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Le Thi","family":"Hoi","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Parasitology, Faculty of Medical Technology, Hanoi Medical University, Hanoi 115600, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6491-2808","authenticated-orcid":false,"given":"Nguyen Vu","family":"Trung","sequence":"additional","affiliation":[{"name":"Pasteur Institute of Ho Chi Minh City, 167 Pasteur Street, District 3, Ho Chi Minh City 722700, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4673-9084","authenticated-orcid":false,"given":"Eli","family":"Schwartz","sequence":"additional","affiliation":[{"name":"The Center for Geographic Medicine, Chaim Sheba Medical Centre, Tel HaShomer, Ramat Gan 5262000, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4091-0493","authenticated-orcid":false,"given":"Amos","family":"Danielli","sequence":"additional","affiliation":[{"name":"Faculty of Engineering, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Max and Anna 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