Development and Validation of a Double‐Antibody Sandwich <scp>ELISA</scp> ( <scp>DAS</scp> ‐ <scp>ELISA</scp> ) for Detection of Anguillid Herpesvirus

Anguillid herpesvirus (AngHV) is a highly pathogenic agent that causes "Mucus sloughing and hemorrhagic septicemia disease" in eels, resulting in high morbidity and cumulative mortality rates. Therefore, accurate diagnosis of AngHV infection is essential for effective clinical management and epidemiological control. In this study, three monoclonal antibodies (mAbs) against AngHV were developed, and their specificity and affinity were validated using Western blotting and indirect immunofluorescence assays. Subsequently, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established for the detection of AngHV, employing mAb 10G8-9F4 as the capture antibody and rabbit polyclonal antibody 9NA (which targets AngHV-ORF95) as the detection antibody. Specificity testing indicated no cross-reactivity with other common aquatic viruses, including Koi herpesvirus (KHV), Grass carp reovirus (GCRV), Infectious pancreatic necrosis virus (IPNV), and Large yellow croaker iridovirus (LYCI). The developed assay effectively identified AngHV in various tissues of infected eels, including the heart, liver, spleen, kidney, gills, intestines, muscle, skin mucus, and fins. Compared to quantitative real-time PCR (qPCR), the detection limit of this assay was determined to be 10,000 copies of AngHV, with an 88.89% concordance rate observed in the analysis of clinical samples between the results of DAS-ELISA and qPCR, thereby demonstrating the reliability of this immunoassay for detecting AngHV in field samples. Overall, the DAS-ELISA developed in this study demonstrates high reactivity and specificity, serving as a rapid and effective diagnostic tool for the detection and prevention of AngHV in eel populations.