Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein
Bovine Coronavirus (BCoV) is a significant pathogen responsible for neonatal calf diarrhea, winter dysentery in adult cattle, and bovine respiratory diseases. Infection with the virus can result in hemorrhagic diarrhea, decreased milk production, and potentially fatal outcomes in cattle, leading to...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2025-02-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2025.1530870/full |
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| author | Qiang Liu Xiaoxia Niu Lingling Jiang Gang Zhang Pu Wang Sinong Zhang Weifeng Gao Huichen Guo Huichen Guo Yujiong Wang Yong Li |
| author_facet | Qiang Liu Xiaoxia Niu Lingling Jiang Gang Zhang Pu Wang Sinong Zhang Weifeng Gao Huichen Guo Huichen Guo Yujiong Wang Yong Li |
| author_sort | Qiang Liu |
| collection | DOAJ |
| description | Bovine Coronavirus (BCoV) is a significant pathogen responsible for neonatal calf diarrhea, winter dysentery in adult cattle, and bovine respiratory diseases. Infection with the virus can result in hemorrhagic diarrhea, decreased milk production, and potentially fatal outcomes in cattle, leading to considerable economic repercussions for the cattle industry. Efficient management of BCoV relies on swift and precise detection techniques. CHO cells were utilized to express a secreted recombinant nucleocapsid protein (N), whereby rabbit polyclonal antibodies (pAb) were generated through immunization. An indirect enzyme-linked immunosorbent assay (iELISA) based on N protein was established for the detection of BCoV antibodies. Reaction conditions were optimized using a checkerboard approach, with the optimal antigen concentration at 1.25 μg/mL and the optimal antibody dilution at 1:200, the cutoff value distinguishing negative and positive serum samples was 0.986. The sensitivity test indicated that this rabbit pAb had a maximum dilution of 218 within the assay range, did not cross-react with BHV-1, BVDV, BRV, and BRSV positive serum samples, and shown great specificity. The developed iELISA method and commercial kit were used to test 58 bovine serum samples, and the concordance rate was 94.83%. In summary, we have developed a cost-efficient and precise iELISA method based on N protein that serves as a useful diagnostic tool for BCoV in clinical samples and epidemiological research. |
| format | Article |
| id | doaj-art-ff34bfaeaaa34b34bc4670be10bf4f9a |
| institution | Kabale University |
| issn | 2297-1769 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj-art-ff34bfaeaaa34b34bc4670be10bf4f9a2025-02-05T07:32:32ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692025-02-011210.3389/fvets.2025.15308701530870Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N proteinQiang Liu0Xiaoxia Niu1Lingling Jiang2Gang Zhang3Pu Wang4Sinong Zhang5Weifeng Gao6Huichen Guo7Huichen Guo8Yujiong Wang9Yong Li10Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaState Key Laboratory of Veterinary Etiological Biology, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaKey Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan, ChinaBovine Coronavirus (BCoV) is a significant pathogen responsible for neonatal calf diarrhea, winter dysentery in adult cattle, and bovine respiratory diseases. Infection with the virus can result in hemorrhagic diarrhea, decreased milk production, and potentially fatal outcomes in cattle, leading to considerable economic repercussions for the cattle industry. Efficient management of BCoV relies on swift and precise detection techniques. CHO cells were utilized to express a secreted recombinant nucleocapsid protein (N), whereby rabbit polyclonal antibodies (pAb) were generated through immunization. An indirect enzyme-linked immunosorbent assay (iELISA) based on N protein was established for the detection of BCoV antibodies. Reaction conditions were optimized using a checkerboard approach, with the optimal antigen concentration at 1.25 μg/mL and the optimal antibody dilution at 1:200, the cutoff value distinguishing negative and positive serum samples was 0.986. The sensitivity test indicated that this rabbit pAb had a maximum dilution of 218 within the assay range, did not cross-react with BHV-1, BVDV, BRV, and BRSV positive serum samples, and shown great specificity. The developed iELISA method and commercial kit were used to test 58 bovine serum samples, and the concordance rate was 94.83%. In summary, we have developed a cost-efficient and precise iELISA method based on N protein that serves as a useful diagnostic tool for BCoV in clinical samples and epidemiological research.https://www.frontiersin.org/articles/10.3389/fvets.2025.1530870/fullbovine coronavirusN proteineukaryotic expressionindirect ELISAantibody detection |
| spellingShingle | Qiang Liu Xiaoxia Niu Lingling Jiang Gang Zhang Pu Wang Sinong Zhang Weifeng Gao Huichen Guo Huichen Guo Yujiong Wang Yong Li Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein Frontiers in Veterinary Science bovine coronavirus N protein eukaryotic expression indirect ELISA antibody detection |
| title | Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein |
| title_full | Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein |
| title_fullStr | Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein |
| title_full_unstemmed | Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein |
| title_short | Establishment of an indirect ELISA method for detecting bovine coronavirus antibodies based on N protein |
| title_sort | establishment of an indirect elisa method for detecting bovine coronavirus antibodies based on n protein |
| topic | bovine coronavirus N protein eukaryotic expression indirect ELISA antibody detection |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2025.1530870/full |
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