Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses
At least three betacoronaviruses have spilled over from bats to humans and caused severe diseases, highlighting the threat of zoonotic transmission. Thus, it is important to enhance surveillance capabilities by developing tools capable of detecting a broad spectrum of bat-borne betacoronaviruses. Th...
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2025-06-01
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| Online Access: | https://www.mdpi.com/1999-4915/17/7/886 |
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| author | Kong Yen Liew Yaju Wang Sneha Sree Mullapudi Dinah binte Aziz Wenjie Fan Min Luo Paul Anantharajah Tambyah Yee-Joo Tan |
| author_facet | Kong Yen Liew Yaju Wang Sneha Sree Mullapudi Dinah binte Aziz Wenjie Fan Min Luo Paul Anantharajah Tambyah Yee-Joo Tan |
| author_sort | Kong Yen Liew |
| collection | DOAJ |
| description | At least three betacoronaviruses have spilled over from bats to humans and caused severe diseases, highlighting the threat of zoonotic transmission. Thus, it is important to enhance surveillance capabilities by developing tools capable of detecting a broad spectrum of bat-borne betacoronaviruses. Three monoclonal antibodies (mAbs) targeting the nucleocapsid (N) protein were generated using recombinant N proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The cross-reactivities of these mAbs were evaluated against a panel of betacoronaviruses. Sandwich ELISAs (sELISAs) were subsequently developed to detect bat-borne betacoronaviruses that have high zoonotic potential. Among the mAbs, 7A7 demonstrated the broadest cross-reactivity, recognizing betacoronaviruses from the <i>Sarbecovirus</i>, <i>Merbecovirus</i> and <i>Hibecovirus</i> subgenera. The first sELISA, based on mAbs 7A7 and 6G10, successfully detected N protein in all clinical swab samples from COVID-19 patients with cycle threshold (Ct) values < 25, achieving 75% positivity overall (12/16). Using this as a reference, a second sELISA was established by pairing mAb 7A7 with mAb 8E2, which binds to multiple merbecoviruses. This assay detected the N protein of two merbecoviruses, namely the human MERS-CoV and bat-borne HKU5-CoV, at high sensitivity and has a limit of detection (LOD) that is comparable to the first sELISA used successfully to detect COVID-19 infection. These broadly reactive mAbs could be further developed into rapid antigen detection kits for surveillance in high-risk populations with close contact with wild bats to facilitate the early detection of potential zoonotic spillover events. |
| format | Article |
| id | doaj-art-9e6dc63b0eca4ff18a4524fb3a382e48 |
| institution | Kabale University |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj-art-9e6dc63b0eca4ff18a4524fb3a382e482025-08-20T03:56:49ZengMDPI AGViruses1999-49152025-06-0117788610.3390/v17070886Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne MerbecovirusesKong Yen Liew0Yaju Wang1Sneha Sree Mullapudi2Dinah binte Aziz3Wenjie Fan4Min Luo5Paul Anantharajah Tambyah6Yee-Joo Tan7Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeInfectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeInfectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeInfectious Diseases Translational Research Programme and Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeDepartment of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117558, SingaporeDepartment of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117558, SingaporeInfectious Diseases Translational Research Programme and Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeInfectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, SingaporeAt least three betacoronaviruses have spilled over from bats to humans and caused severe diseases, highlighting the threat of zoonotic transmission. Thus, it is important to enhance surveillance capabilities by developing tools capable of detecting a broad spectrum of bat-borne betacoronaviruses. Three monoclonal antibodies (mAbs) targeting the nucleocapsid (N) protein were generated using recombinant N proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The cross-reactivities of these mAbs were evaluated against a panel of betacoronaviruses. Sandwich ELISAs (sELISAs) were subsequently developed to detect bat-borne betacoronaviruses that have high zoonotic potential. Among the mAbs, 7A7 demonstrated the broadest cross-reactivity, recognizing betacoronaviruses from the <i>Sarbecovirus</i>, <i>Merbecovirus</i> and <i>Hibecovirus</i> subgenera. The first sELISA, based on mAbs 7A7 and 6G10, successfully detected N protein in all clinical swab samples from COVID-19 patients with cycle threshold (Ct) values < 25, achieving 75% positivity overall (12/16). Using this as a reference, a second sELISA was established by pairing mAb 7A7 with mAb 8E2, which binds to multiple merbecoviruses. This assay detected the N protein of two merbecoviruses, namely the human MERS-CoV and bat-borne HKU5-CoV, at high sensitivity and has a limit of detection (LOD) that is comparable to the first sELISA used successfully to detect COVID-19 infection. These broadly reactive mAbs could be further developed into rapid antigen detection kits for surveillance in high-risk populations with close contact with wild bats to facilitate the early detection of potential zoonotic spillover events.https://www.mdpi.com/1999-4915/17/7/886betacoronavirusnucleocapsid proteinmonoclonal antibodysandwich ELISAbat-borne viruseszoonotic surveillance |
| spellingShingle | Kong Yen Liew Yaju Wang Sneha Sree Mullapudi Dinah binte Aziz Wenjie Fan Min Luo Paul Anantharajah Tambyah Yee-Joo Tan Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses Viruses betacoronavirus nucleocapsid protein monoclonal antibody sandwich ELISA bat-borne viruses zoonotic surveillance |
| title | Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses |
| title_full | Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses |
| title_fullStr | Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses |
| title_full_unstemmed | Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses |
| title_short | Designing Sandwich ELISA with Broadly Reactive Anti-Nucleocapsid Monoclonal Antibodies to Detect Bat-Borne Merbecoviruses |
| title_sort | designing sandwich elisa with broadly reactive anti nucleocapsid monoclonal antibodies to detect bat borne merbecoviruses |
| topic | betacoronavirus nucleocapsid protein monoclonal antibody sandwich ELISA bat-borne viruses zoonotic surveillance |
| url | https://www.mdpi.com/1999-4915/17/7/886 |
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