Optimizing ovine brucellosis serodiagnosis: evaluation of recombinant Brucella antigens and multi-antigen combinations for iELISA

Background Brucellosis, a major zoonosis caused by Brucella spp., affects both animals and humans. Accurate serological diagnosis is crucial for effective disease control, yet current methods often lack optimal sensitivity and specificity. This study aimed to evaluate four recombinant Brucella antigens (rOmp19, rOmp2b, rOmp31, and rBP26) as diagnostic antigens, both individually and in combination, to identify an optimal multi-antigen cocktail for an improved indirect ELISA (iELISA). Methods Four recombinant Brucella antigens were expressed in E. coli and purified by Ni-NTA affinity chromatography. A total of 224 sheep serum samples without a history of brucellosis vaccination were employed. The samples were classified as reference-positive ( n = 20) or reference-negative ( n = 204) based on parallel testing with a commercial cELISA kit and the serum agglutination test (SAT), following relevant WOAH recommendations. iELISA conditions were optimized by checkerboard titration. Diagnostic performance was assessed by calculating sensitivity, specificity, accuracy, and the area under the receiver operating characteristic curve (AUC). Results Evaluation of single antigens revealed that rOmp19 had 100% sensitivity (20/20; 95% CI: 83.2%–100%) and the highest AUC (0.9931), but moderate specificity (93.62%; 95% CI: 89.1%–96.5%). Conversely, rOmp2b, rOmp31, and rBP26 each showed 100% specificity (95% CI: 98.2%–100%) with lower sensitivity (85%; 95% CI: 62.1%–96.8%) and AUCs (0.9547, 0.9498, and 0.9645). Multi-antigen formulations improved overall diagnostic balance. The two-antigen combination (rOmp19 + rBP26) increased specificity to 98.04% (95% CI: 95.0%–99.5%) while maintaining 90.0% sensitivity (95% CI: 68.3%–98.8%; AUC = 0.9841). The three-antigen cocktail (rOmp19 + rOmp31 + rBP26) achieved the optimal profile, retaining 100% sensitivity, elevating specificity to 99.51% (95% CI: 97.3%–99.9%), and yielding the best AUC among the multi-antigen combinations (AUC = 0.9850). Notably, adding a fourth antigen (rOmp19 + rOmp2b + rOmp31 + rBP26) substantially impaired performance, reducing sensitivity to 70%, specificity to 92.65%, and AUC to 0.8711. Conclusion The three-antigen combination (rOmp19 + rOmp31 + rBP26) identified in this study achieves an optimal balance between sensitivity and specificity, supporting its potential to enhance the performance of brucellosis serodiagnosis. This work provides experimental evidence and constitutes an important step toward the development of multi-antigen iELISA-based brucellosis detection kits. However, further validation and optimization are required before clinical or field application.