tetano
Editor, Senior Moderator
Pathogens
. 2025 Jun 19;14(6):605.
doi: 10.3390/pathogens14060605. Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses
Kaixin Li[SUP] 1 [/SUP], Misa Katayama[SUP] 1 [/SUP], Ayano Ichikawa[SUP] 1 [/SUP], Hiromichi Matsugo[SUP] 1 2 [/SUP], Yuta Wakabayashi[SUP] 1 [/SUP], Akiko Takenaka-Uema[SUP] 1 [/SUP], Wataru Sekine[SUP] 1 [/SUP], Taisuke Horimoto[SUP] 1 [/SUP], Shin Murakami[SUP] 1 [/SUP]
Affiliations
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-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.
Keywords: antigen detection; bat merbecoviruses; monoclonal antibody; sandwich ELISA.
. 2025 Jun 19;14(6):605.
doi: 10.3390/pathogens14060605. Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses
Kaixin Li[SUP] 1 [/SUP], Misa Katayama[SUP] 1 [/SUP], Ayano Ichikawa[SUP] 1 [/SUP], Hiromichi Matsugo[SUP] 1 2 [/SUP], Yuta Wakabayashi[SUP] 1 [/SUP], Akiko Takenaka-Uema[SUP] 1 [/SUP], Wataru Sekine[SUP] 1 [/SUP], Taisuke Horimoto[SUP] 1 [/SUP], Shin Murakami[SUP] 1 [/SUP]
Affiliations
- PMID: 40559613
- PMCID: PMC12196402
- DOI: 10.3390/pathogens14060605
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-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.
Keywords: antigen detection; bat merbecoviruses; monoclonal antibody; sandwich ELISA.