Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant.
Abstract: Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference standard. The detection limit for the Bucyrus EAV strain was 36 PFU, and the method had a good linear relationship between 72-2297 PFU. The AC-ELISA could specifically detect the Bucyrus EAV strain and had no cross-reaction with other equine viruses. The sensitivity of the AC-ELISA was much higher than that of a western blotting assay but lower than that of a real-time PCR method. However, as a quantitative antigen detection method, the sensitivity of the AC-ELISA was approximately 300 times than the western blotting assay. Furthermore, the AC-ELISA assay could be successfully used in quantification of viral content in an in vitro infection assay, such as a one-step growth curve of EAV, as well as in a transfection assay, such as virus rescue from an infectious cDNA clone of EAV. These results show that the AC-ELISA established in this study is a good alternative for antigen detection of EAV, being a simple, convenient and quantitative detection method for EAV antigens.
Publication Date: 2018-02-12 PubMed ID: 29435711DOI: 10.1007/s00705-018-3746-5Google Scholar: Lookup
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- Journal Article
- Antibodies
- Antigen
- Comparative Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Equine Diseases
- Equine Health
- Equine Viral Arteritis
- Horses
- Immunology
- In Vitro Research
- Infectious Disease
- Laboratory Methods
- Monoclonal Antibodies
- Real-Time PCR
- Veterinary Medicine
- Virology
- Virus
- Western Blot
Summary
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The researchers developed a sensitive and quick method to quantify equine arteritis virus (EAV) using an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). The method proved very effective and was successful in measuring the viral content, showing a good alternative for antigen detection of EAV.
Methodology
- The researchers established a quantitative detection method for EAV known as an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA).
- This was achieved by employing two EAV nucleoprotein monoclonal antibodies, referred to as 2B9 and 2B3, which were developed in this study.
- Different conditions were examined to optimize this method, resulting in the decision to use mAb 2B9 as the capture antibody, while HRP-labeled 2B3 was chosen as the detecting antibody.
Results and Effectiveness
- The AC-ELISA demonstrated a strong standard curve when viral particles from the Bucyrus EAV strain were used as the reference standard.
- The method had a detection limit of 36 PFU for the Bucyrus EAV strain, and a strong linear relationship was observed between the concentration ranges of 72-2297 PFU.
- The AC-ELISA could specifically detect the Bucyrus EAV strain with no cross-reaction with other equine viruses.
Comparison with Other Methods
- Compared to a western blotting assay, the sensitivity of the AC-ELISA was significantly higher, around 300 times.
- However, when compared to a real-time PCR method, the AC-ELISA was found to be less sensitive.
Practical uses of AC-ELISA
- The AC-ELISA assay was successfully applied to quantify viral content in in vitro infection assays such as the one-step growth curve of EAV.
- It was also successful in transfection assays like virus rescue from an infectious cDNA clone of EAV.
- The results indicated that the AC-ELISA established in this study is a good alternative for EAV antigen detection, being convenient, simple, and capable of providing quantitative measurements.
Cite This Article
APA
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.
(2018).
Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant.
Arch Virol, 163(6), 1469-1478.
https://doi.org/10.1007/s00705-018-3746-5 Publication
Researcher Affiliations
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Road, Xiangfang District, Harbin, 150069, People's Republic of China.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Road, Xiangfang District, Harbin, 150069, People's Republic of China.
- Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, College of Veterinary Medicine, Inner Mongolia Agricultural University, Ministry of Agriculture, Hohhot, 010018, China.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Road, Xiangfang District, Harbin, 150069, People's Republic of China.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Road, Xiangfang District, Harbin, 150069, People's Republic of China.
- Virology Unit, Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland.
- Virology Unit, Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland.
- Virology Unit, Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Road, Xiangfang District, Harbin, 150069, People's Republic of China. xjw@hvri.ac.cn.
MeSH Terms
- Animals
- Antibodies, Monoclonal / biosynthesis
- Antibodies, Monoclonal / chemistry
- Antibodies, Monoclonal / isolation & purification
- Antibodies, Viral / biosynthesis
- Antibodies, Viral / chemistry
- Antibodies, Viral / isolation & purification
- Antigens, Viral / analysis
- Antigens, Viral / genetics
- Antigens, Viral / immunology
- Arterivirus Infections / diagnosis
- Arterivirus Infections / veterinary
- Arterivirus Infections / virology
- Blotting, Western
- Cell Line
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / standards
- Enzyme-Linked Immunosorbent Assay / veterinary
- Epithelial Cells
- Equartevirus / genetics
- Equartevirus / immunology
- Equartevirus / isolation & purification
- Female
- HEK293 Cells
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horseradish Peroxidase / chemistry
- Horses
- Humans
- Immunization
- Limit of Detection
- Mice
- Mice, Inbred BALB C
- Virion / genetics
- Virion / immunology
Grant Funding
- 31402203 / National Natural Science Foundation of China
- C2017080 / Natural Science Foundation of Heilongjiang Province of China
Citations
This article has been cited 1 times.- Bu J, Deng Z, Liu H, Li J, Wang D, Yang Y, Zhong S. Current methods and prospects of coronavirus detection. Talanta 2021 Apr 1;225:121977.
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