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Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant

Author:
Qi, Ting, Hu, Yue, Hu, Zhe, Zhao, Shihua, Cullinane, Ann, Lyons, Pamela, Gildea, Sarah, Wang, Xiaojun
Source:
Archives of virology 2018 v.163 no.6 pp. 1469-1478
ISSN:
0304-8608
Subject:
Equine arteritis virus, Western blotting, antigen detection, antigens, complementary DNA, cross reaction, detection limit, enzyme-linked immunosorbent assay, growth curves, horses, monoclonal antibodies, nucleoproteins, quantitative polymerase chain reaction, reference standards, transfection, virion, viruses
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.
Agid:
5964933