The establishment of a universal blocking ELISA for echinococcosis using immunoglobulin against a new conserved linear B-cell epitope.

Abstract

Echinococcosis is a neglected tropical disease that poses a severe threat to global public health and socioeconomic development. Given its alarming global spread, there is an urgent need to develop accurate diagnostic methods to support targeted and personalized treatment. Echinococcus multilocularis Em18 has been demonstrated to exhibit the most prominent diagnostic relevance in the progression of alveolar echinococcosis (AE). In this study, six highly specific monoclonal antibodies (mAbs) against the Em18 protein-designated 8G6F, 11E2E, 11E7C, 11E7E, 11E8C, and 11E10D-were generated using hybridoma technology. Indirect ELISA showed that the supernatant titers ranged from 1:1,600 to 1:12,800, while the ascites titers varied from 1:128,000 to 1:512,000. Further analysis indicated that all mAbs belonged to the IgG1 κ isotype. After purification, the ascites-derived mAbs exhibited expected heavy-chain and light-chain bands at approximately 50 kDa and 25 kDa, respectively. Notably, western blot and indirect immunofluorescence assay collectively demonstrated that all mAbs exhibited high specificity for recombinant Em18 protein of approximately 18.65 kDa. Through peptide scanning technology, we identified the antigenic epitope recognized by six mAbs as the region ⁹⁹RMREKHDAKHKS¹⁰⁸, a highly conserved linear B-cell epitope on the Em18 protein. Amino acid sequence alignment confirmed the complete conservation of this epitope, which is surface-exposed. Furthermore, the optimal blocking antibody 11E8C was selected and labeled with horseradish peroxidase (HRP) based on which a blocking ELISA was established using the anti-Em18 mAbs. Serum samples with a percent inhibition (PI) ≥22.48% were determined as positive, and those with PI ≤17.64% as negative. This method showed no cross-reactivity with positive sera against Toxoplasma gondii, Babesia spp., Cysticercus tenuicollis, Coenurus cerebralis, and other non-echinococcosis pathogens. Sensitivity testing showed a positive detection rate of 90%, and positive signals remained stable even when positive sera were diluted up to 1:16. The intra-assay and inter-assay coefficients of variation (CV) were both below 10%. In a concordance evaluation using 47 clinical serum samples from cattle and sheep, the overall agreement between our assay and a commercial kit was 93.62%. Furthermore, the mAbs developed in this study accurately identified intermediate and advanced AE infections, providing a reliable tool for assessing disease progression. Importantly, its cross-reactivity with cystic echinococcosis (CE)-infected sera demonstrates promising potential for the broad-spectrum serodiagnosis of echinococcosis. Collectively, these results suggest that this study provides a novel technical resource for improving serological detection and developing early diagnostic reagents for echinococcosis and offers experimental support for the surveillance, prevention, control, and evaluation of this disease.IMPORTANCEEchinococcosis is a neglected tropical disease posing significant threats to global public health and economic development. We have developed six highly specific mAbs capable of precisely recognizing the Em18 protein, a key biomarker for disease progression, and have for the first time identified their antigenic epitope. These antibodies can not only accurately distinguish intermediate and advanced infection stages but also exhibit cross-reactivity with cystic echinococcosis, demonstrating potential as a broad-spectrum diagnostic tool. This achievement paves a new path for developing rapid and precise clinical detection methods, holding significant importance for improving disease prevention and control capabilities.