A novel indirect ELISA based on BP26 mAb to differentiate Brucella melitensis M5-90BPΔ26 vaccine immunization

Brucellosis, primarily caused by the highly invasive Brucella melitensis (B.melitensis), remains a major global zoonotic threat. Differentiating infected from vaccinated animals (DIVA) is critical for effective surveillance. In this study, we developed an indirect ELISA (iELISA) based on a novel monoclonal antibody (mAb) targeting the BP26 protein. Under optimized conditions, the assay established a cut-off value of 0.4, yielding a diagnostic sensitivity of 96.6% (29/30) and specificity of 100% (30/30). The iELISA demonstrated high analytical sensitivity with no cross-reactivity against common pathogens (E. coli, Salmonella, and Y. enterocolitica). Intra- and inter-assay coefficients of variation (CV) were all <10%, confirming robust reproducibility. Comparative analysis with a commercial kit showed high agreement rates across sera from goats immunized with M5-90BPΔ26 (90.18%), M5-90 (94.29%), and non-immune controls (96.02%). These results suggest that this iELISA is a reliable diagnostic tool for distinguishing wild-type infection from M5-90ΔBP26 vaccination in clinical practice.