Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses
<i>Merbecovirus</i>, a subgenus of <i>Betacoronavirus</i>, 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. I...
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2025-06-01
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| author | Kaixin Li Misa Katayama Ayano Ichikawa Hiromichi Matsugo Yuta Wakabayashi Akiko Takenaka-Uema Wataru Sekine Taisuke Horimoto Shin Murakami |
| author_facet | Kaixin Li Misa Katayama Ayano Ichikawa Hiromichi Matsugo Yuta Wakabayashi Akiko Takenaka-Uema Wataru Sekine Taisuke Horimoto Shin Murakami |
| author_sort | Kaixin Li |
| collection | DOAJ |
| description | <i>Merbecovirus</i>, a subgenus of <i>Betacoronavirus</i>, 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—VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5—with 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 × 10<sup>3</sup> 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. |
| format | Article |
| id | doaj-art-e6bb3e315ea6444d9dec24cf4fabe120 |
| institution | Kabale University |
| issn | 2076-0817 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj-art-e6bb3e315ea6444d9dec24cf4fabe1202025-08-20T03:27:36ZengMDPI AGPathogens2076-08172025-06-0114660510.3390/pathogens14060605Establishment of a Sandwich ELISA for Detection of Pan-MerbecovirusesKaixin Li0Misa Katayama1Ayano Ichikawa2Hiromichi Matsugo3Yuta Wakabayashi4Akiko Takenaka-Uema5Wataru Sekine6Taisuke Horimoto7Shin Murakami8Laboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, JapanLaboratory of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan<i>Merbecovirus</i>, a subgenus of <i>Betacoronavirus</i>, 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—VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5—with 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 × 10<sup>3</sup> 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.https://www.mdpi.com/2076-0817/14/6/605bat merbecovirusessandwich ELISAantigen detectionmonoclonal antibody |
| spellingShingle | Kaixin Li Misa Katayama Ayano Ichikawa Hiromichi Matsugo Yuta Wakabayashi Akiko Takenaka-Uema Wataru Sekine Taisuke Horimoto Shin Murakami Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses Pathogens bat merbecoviruses sandwich ELISA antigen detection monoclonal antibody |
| title | Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses |
| title_full | Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses |
| title_fullStr | Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses |
| title_full_unstemmed | Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses |
| title_short | Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses |
| title_sort | establishment of a sandwich elisa for detection of pan merbecoviruses |
| topic | bat merbecoviruses sandwich ELISA antigen detection monoclonal antibody |
| url | https://www.mdpi.com/2076-0817/14/6/605 |
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