Hepatitis E virus (HEV) is a significant zoonotic pathogen posing a global threat to both human and animal health. To advance broad-spectrum detection strategies for HEV, we developed high-affinity monoclonal antibodies targeting the conserved region of the HEV ORF2 protein and established a blocking ELISA based on these antibodies. Antigen coating concentration, antibody dilution and others conditions were optimized using a checkerboard titration approach. Using well-characterized serum samples, cut-off values for different species were established through ROC curve analysis: 26.0 % for swine sera, achieving 96.0 % sensitivity and specificity; and 35.5 % for Apodemus peninsulae sera, with sensitivity and specificity of 88.0 % and 98.0 %, respectively. The assay exhibited no cross-reactivity with PCV2, PRRSV, and showed a maximum detection dilution twice that of a commercial indirect ELISA kit, indicating superior sensitivity. Clinical validation with 212 swine serum samples demonstrated a concordance rate of 97.64 % with a commercial ELISA kit. Furthermore, the assay effectively monitored the dynamic changes of anti-HEV antibodies in Apodemus peninsulae, with results closely matching those of the commercial kit. This ORF2 epitope based blocking ELISA enables cross genotype and cross species detection, thereby minimizing false negatives due to viral diversity and offering substantial utility for diagnosis, food safety, zoonotic surveillance, and vaccine development.

Keywords: Blocking ELISA; HEV; Monoclonal antibodies; ORF2.