Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen
African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody de...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-09-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/15/9/1939 |
| _version_ | 1797576429153550336 |
|---|---|
| author | Zhi Wu Huipeng Lu Dewei Zhu Jun Xie Fan Sun Yan Xu Hua Zhang Zhijun Wu Wenlong Xia Shanyuan Zhu |
| author_facet | Zhi Wu Huipeng Lu Dewei Zhu Jun Xie Fan Sun Yan Xu Hua Zhang Zhijun Wu Wenlong Xia Shanyuan Zhu |
| author_sort | Zhi Wu |
| collection | DOAJ |
| description | African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in <i>Escherichia coli</i> (<i>E. coli</i>) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential. |
| first_indexed | 2024-03-10T21:51:48Z |
| format | Article |
| id | doaj.art-b5727799472b4702bb309811aa9042f7 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T21:51:48Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-b5727799472b4702bb309811aa9042f72023-11-19T13:23:50ZengMDPI AGViruses1999-49152023-09-01159193910.3390/v15091939Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein AntigenZhi Wu0Huipeng Lu1Dewei Zhu2Jun Xie3Fan Sun4Yan Xu5Hua Zhang6Zhijun Wu7Wenlong Xia8Shanyuan Zhu9Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaJiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaYancheng Engineering Research Center of Animal Biologics, School of Marine and Biological Engineering, Yancheng Teachers University, Yancheng 224007, ChinaJiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaJiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaJiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaSchool of Pharmacy, Yancheng Teachers University, Yancheng 224007, ChinaSchool of Pharmacy, Yancheng Teachers University, Yancheng 224007, ChinaYancheng Engineering Research Center of Animal Biologics, School of Marine and Biological Engineering, Yancheng Teachers University, Yancheng 224007, ChinaJiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaAfrican swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in <i>Escherichia coli</i> (<i>E. coli</i>) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.https://www.mdpi.com/1999-4915/15/9/1939African swine fever virusp15 proteintag-free antigenindirect ELISA |
| spellingShingle | Zhi Wu Huipeng Lu Dewei Zhu Jun Xie Fan Sun Yan Xu Hua Zhang Zhijun Wu Wenlong Xia Shanyuan Zhu Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen Viruses African swine fever virus p15 protein tag-free antigen indirect ELISA |
| title | Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen |
| title_full | Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen |
| title_fullStr | Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen |
| title_full_unstemmed | Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen |
| title_short | Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen |
| title_sort | developing an indirect elisa for the detection of african swine fever virus antibodies using a tag free p15 protein antigen |
| topic | African swine fever virus p15 protein tag-free antigen indirect ELISA |
| url | https://www.mdpi.com/1999-4915/15/9/1939 |
| work_keys_str_mv | AT zhiwu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT huipenglu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT deweizhu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT junxie developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT fansun developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT yanxu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT huazhang developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT zhijunwu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT wenlongxia developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen AT shanyuanzhu developinganindirectelisaforthedetectionofafricanswinefevervirusantibodiesusingatagfreep15proteinantigen |