{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T08:59:08Z","timestamp":1786697948421,"version":"build-2736575974"},"reference-count":27,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,19]],"date-time":"2025-06-19T00:00:00Z","timestamp":1750291200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Agency for Medical Research and Development (AMED)","award":["23fk0108602h"],"award-info":[{"award-number":["23fk0108602h"]}]},{"name":"Japan Agency for Medical Research and Development (AMED)","award":["JP223fa627001"],"award-info":[{"award-number":["JP223fa627001"]}]},{"name":"The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA)","award":["23fk0108602h"],"award-info":[{"award-number":["23fk0108602h"]}]},{"name":"The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA)","award":["JP223fa627001"],"award-info":[{"award-number":["JP223fa627001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Merbecovirus, a subgenus of Betacoronavirus, includes MERS-CoV and multiple bat-derived viruses with zoonotic potential. Given the unpredictable emergence of these viruses and their genetic diversity, development of broad-spectrum diagnostic tools is expected. In this study, we established a sandwich ELISA targeting the nucleocapsid (N) protein of merbecoviruses. We generated monoclonal antibodies (mAbs) using recombinant N protein of a bat merbecovirus, VsCoV-1, and selected cross-reactive clones for other merbecoviruses. Three mAbs showed strong reactivities with multiple merbecoviruses but not with SARS-CoV-2 or endemic human coronaviruses. Pairwise ELISA screening identified 1A8\/10H6 mAbs as the optimal combination for detection of N protein from six merbecoviruses\u2014VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5\u2014with limits of detection (LODs) below 7.81 ng\/mL, including 1.25 ng\/mL for VsCoV-1. Infectious bat merbecovirus EjCoV-3 was detected at 1.3 \u00d7 103 PFU\/mL. No cross-reactivity was observed with non-merbecoviruses, indicating its high specificity. This sandwich ELISA offers a rapid, reproducible, and cost-effective diagnostic platform with potential for high-throughput screening and automation. Moreover, its design is amenable to adaptation into point-of-care formats such as lateral flow assays, highlighting its value for field-based surveillance and pandemic preparedness.<\/jats:p>","DOI":"10.3390\/pathogens14060605","type":"journal-article","created":{"date-parts":[[2025,6,19]],"date-time":"2025-06-19T08:43:58Z","timestamp":1750322638000},"page":"605","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses"],"prefix":"10.3390","volume":"14","author":[{"given":"Kaixin","family":"Li","sequence":"first","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Misa","family":"Katayama","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayano","family":"Ichikawa","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiromichi","family":"Matsugo","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"},{"name":"Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Wakabayashi","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6577-896X","authenticated-orcid":false,"given":"Akiko","family":"Takenaka-Uema","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wataru","family":"Sekine","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9201-9424","authenticated-orcid":false,"given":"Taisuke","family":"Horimoto","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0274-9165","authenticated-orcid":false,"given":"Shin","family":"Murakami","sequence":"additional","affiliation":[{"name":"Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1819","DOI":"10.3201\/eid1911.131172","article-title":"Middle East respiratory syndrome coronavirus in bats, Saudi Arabia","volume":"19","author":"Memish","year":"2013","journal-title":"Emerg. 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