Preparation of monoclonal antibodies against Echinococcus multilocularis CIP2A and development of blocking and sandwich ELISAs.

Alveolar echinococcosis (AE), caused by the metacestode stage of Echinococcus multilocularis, is a highly lethal zoonosis. Its insidious progression frequently precludes diagnosis until advanced stages, and the paucity of effective early detection tools remains a critical impediment to disease control. In this study, we aimed to evaluate the diagnostic utility of EmCIP2A as a target antigen and develop monoclonal antibody-based tools for the serological and non-invasive detection of E. multilocularis infection. Six monoclonal antibodies (mAbs) were generated against EmCIP2A, a germinative cell-specific marker of the parasite. Isotyping identified 1F3C8 and 2F7G3 as IgG1, and 1G9E4, 3F1G4, 4B1C6, and 5A7C11 as IgG2b, all bearing kappa light chains. Western blotting and indirect immunofluorescence confirmed that all mAbs specifically recognize both recombinant and native EmCIP2A. Four linear B-cell epitopes were delineated with 1F3C8/5A7C11 targeted 229MADLSLTEER238, 4B1C6 bound 384HHSEATLNRL393, 2F7G3 recognized 432AEVEAKREAL441, and 1G9E4/3F1G4 reacted with 574TGNRVQPANA583. Based on epitope and affinity screening, a blocking ELISA employing HRP-1F3C8 was established (AUC = 0.8494; sensitivity, 90.63%; specificity, 72.73%). In addition, a double-antibody sandwich ELISA using 1F3C8 as capture antibody and HRP-2F7G3 as detection antibody was further developed, displaying excellent linearity (1-1000 ng/mL, R2 = 0.9973); preliminary fecal testing yielded detection rates of 70% in infected mice and 50% in infected dogs, confirming its utility for active infection surveillance across intermediate and definitive hosts. Collectively, the two ELISA systems provide complementary modalities, serological screening and non-invasive fecal antigen detection, which together expand the diagnostic repertoire against E. multilocularis infection.