Raw data for I-ELISA.

BackgroundBrucellosis is a widespread zoonotic disease with approximately 2.1 million new human cases annually. Traditional serological methods, which rely on lipopolysaccharide (LPS) as a diagnostic antigen, suffer from cross-reactivity with other Gram-negative bacteria. To address these issues, we aimed to develop a multiepitope fusion protein for improved brucellosis diagnosis.MethodsWe identified linear B-cell epitopes from Brucella proteins using the Immune Epitope Database (IEDB) and constructed a multiepitope fusion protein. The fusion protein was expressed through prokaryotic expression, purified, and was evaluated using an indirect enzyme-linked immunosorbent assay (iELISA) with serum samples from patients with confirmed brucellosis (n = 279) and negative controls from febrile patients (n = 126). To rigorously assess cross-reactivity, it was also tested against a separate panel of sera from 283 non-brucellosis febrile patients with laboratory-confirmed infections by other bacterial pathogens.ResultsThe engineered fusion protein, consisting of 11 optimized linear B-cell epitopes derived from the consolidation of 23 initial epitopes obtained from the IEDB, was assessed using iELISA. This evaluation yielded an area under the curve (AUC) of 0.9912 relative to LPS, demonstrating a sensitivity of 95.34% and a specificity of 93.65% in comparison to the negative control group (n = 126). Critically, when tested against the distinct cross-reactivity panel, the fusion protein exhibited cross-reactivity with 9 serum samples from 283 patients infected with other bacterial pathogens, whereas LPS showed cross-reactivity in 41 out of 283 samples.ConclusionsWhen evaluated against a clinically relevant control cohort of non-brucellosis febrile patients, the fusion protein demonstrated a substantial advantage by exhibiting significantly lower cross-reactivity compared to LPS, which frequently cross-reacted with other bacterial infections. The multiepitope fusion protein developed in this study demonstrates significant potential as a diagnostic tool for brucellosis. However, the study’s limitations, including a small sample size and lack of information on Brucella species, suggest the need for further research with larger and more diverse sample sets to fully validate its clinical application.