Development and Application of an Antigen Capture ELISA for the Detection of Enzootic Nasal Tumor Virus-2

Enzootic nasal tumor virus (ENTV) is the etiological agent responsible for enzootic nasal adenocarcinoma (ENA), a chronic and contagious disease predominantly affecting sheep and goats. ENTV is classified into two distinct types: ENTV-1, which infects sheep, and ENTV-2, which infects goats. ENA has been globally reported in small ruminant-rearing regions, causing significant mortality and substantial economic impacts in affected flocks. There is currently no standardized detection method for ENA. In this study, we successfully generated a monoclonal antibody (mAb) and a polyclonal antibody (pAb) against the ENTV-2 capsid protein (p27), and identified the epitope of the mAb, which was found to be highly conserved among different ENTV-2 isolates. An antigen capture ELISA (acELISA) was then successfully developed using the mAb as the capture antibody and the pAb as the detection antibody to specifically detect p27 of ENTV-2 in nasal secretions. The cut-off value of the acELISA was determined to be 0.1052 by analyzing S/P values. The detection limit of this assay was 0.16 ng/mL of rp27 protein and equivalent to 844 copies/μL of ENTV-2 RNA. Specificity tests showed that the method had no cross-reaction with other prevalent small ruminant pathogens. The coincidence rates of the developed acELISA compared with western blotting and qRT-PCR assays were 98.95% (189/191) and 96.34% (184/191), respectively. Furthermore, the acELISA was applied to assess ENTV-2 in 1228 clinical nasal swab samples collected from seven provinces in China. The results demonstrated that the positivity rate varied between 0.00% and 28.21%. In conclusion, we successfully developed an acELISA with high specificity, sensitivity and reproducibility for the detection of ENTV-2 antigen. This high-throughput method for the detection of ENTV-2 represents a significant advancement in the field and may contribute to the prevention and control of ENTV-2.