Development of monoclonal antibodies against goose astrovirus 2 ORF2 protein and establishment of an indirect competitive ELISA detection method.

Gosling gout, caused by goose astrovirus 2 (GAstV-2), poses a significant threat to the goose industry due to its high morbidity and mortality, while effective control measures remain unavailable. The development of specific countermeasures is hindered by a limited characterization of monoclonal antibodies (mAbs) and epitopes against the major ORF2 antigen. To address this, we generated two mAbs (A12G1 and A10D12) against GAstV-2 ORF2 using hybridoma technology. Epitope mapping identified two novel linear B-cell epitopes, 1MADRA5 (A12G1) and 36YKPQKLPMKA45 (A10D12), residing in the conserved S domain. These epitopes were highly conserved among GAstV-2 isolates but demonstrated notable divergence from other avian astroviruses. The specific reactivity of both mAbs with GAstV-2 was confirmed by Western blotting, immunofluorescence, and immunohistochemistry. Furthermore, leveraging mAb A10D12, we established a highly sensitive and specific indirect competitive ELISA (icELISA) for detecting GAstV-2 antibodies in goose serum. The icELISA demonstrated excellent reproducibility, high sensitivity, and no cross-reactivity with antisera against other common waterfowl pathogens. This study not only expands the known epitope repertoire of GAstV-2 ORF2 but also provides valuable mAbs and a robust serological tool for functional studies of ORF2 and for monitoring GAstV-2 infections.