Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus
ABSTRACT: Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish pero...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | Poultry Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0032579122006204 |
| _version_ | 1797987157583855616 |
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| author | Tianxiang Chen Baoyuan Liu Yiyang Chen Xueting Wang Meimei Zhang Xukun Dang Qin Zhao En-Min Zhou |
| author_facet | Tianxiang Chen Baoyuan Liu Yiyang Chen Xueting Wang Meimei Zhang Xukun Dang Qin Zhao En-Min Zhou |
| author_sort | Tianxiang Chen |
| collection | DOAJ |
| description | ABSTRACT: Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection. |
| first_indexed | 2024-04-11T07:44:11Z |
| format | Article |
| id | doaj.art-ac9451d4d2df41e5bb0f712ebd3c36a2 |
| institution | Directory Open Access Journal |
| issn | 0032-5791 |
| language | English |
| last_indexed | 2024-04-11T07:44:11Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Poultry Science |
| spelling | doaj.art-ac9451d4d2df41e5bb0f712ebd3c36a22022-12-22T04:36:23ZengElsevierPoultry Science0032-57912023-01-011021102326Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virusTianxiang Chen0Baoyuan Liu1Yiyang Chen2Xueting Wang3Meimei Zhang4Xukun Dang5Qin Zhao6En-Min Zhou7Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, Shaanxi, China; Corresponding author:Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, Shaanxi, ChinaDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, Shaanxi, ChinaABSTRACT: Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection.http://www.sciencedirect.com/science/article/pii/S0032579122006204nanobody-HRP fusion proteinHEK293Tcompetitive ELISAavian HEVantibody detection |
| spellingShingle | Tianxiang Chen Baoyuan Liu Yiyang Chen Xueting Wang Meimei Zhang Xukun Dang Qin Zhao En-Min Zhou Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus Poultry Science nanobody-HRP fusion protein HEK293T competitive ELISA avian HEV antibody detection |
| title | Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus |
| title_full | Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus |
| title_fullStr | Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus |
| title_full_unstemmed | Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus |
| title_short | Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus |
| title_sort | development of a novel competitive elisa based on nanobody horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis e virus |
| topic | nanobody-HRP fusion protein HEK293T competitive ELISA avian HEV antibody detection |
| url | http://www.sciencedirect.com/science/article/pii/S0032579122006204 |
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