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

Ting Qi, Yue Hu, Zhe Hu, Shihua Zhao, Ann Cullinane, Pamela Lyons, Sarah Gildea, Xiaojun Wang

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.