Conserved surface-exposed linear epitopes of Echinococcus multilocularis thioredoxin peroxidase enable non-invasive diagnosis of alveolar echinococcosis in intermediate host.

Alveolar echinococcosis (AE) is a severe zoonotic parasitic disease caused by the larval stage of Echinococcus multilocularis, for which early and accurate diagnosis remains challenging. In this study, we targeted E. multilocularis thioredoxin peroxidase (EmTPx), a highly abundant excretory/secretory protein conserved across the genus Echinococcus, for diagnostic development. Monoclonal antibodies (mAbs) against EmTPx were generated, and two linear B‑cell epitopes, 174HPGSK178 and 189SFMSS193, were identified. Both epitopes are strictly conserved within Echinococcus but markedly divergent from mammalian host sequences. Structural modeling revealed that the epitopes are surface‑exposed, with 174HPGSK178 located in a flexible loop and 189SFMSS193 adjacent to an α‑helix, conformations favorable for antibody recognition. Two ELISA methods were established using the epitope‑specific mAbs. The blocking ELISA showed moderate diagnostic performance. More importantly, the sandwich ELISA enabled direct detection of EmTPx antigen in fecal samples of intermediate hosts, revealing a detection rate of 85% in mouse feces at an early infection stage. With a linear range of 0-250 ng/mL, a cutoff of 0.1724, and intra‑ and inter‑assay precision below 10%, this sandwich ELISA demonstrated robust analytical performance. In conclusion, this proof-of-concept study identifies two evolutionarily conserved and structurally exposed EmTPx epitopes and demonstrates their potential utility for the development of monoclonal antibody-based approaches for non-invasive detection of echinococcosis.