ArticleFrontiers in cellular and infection microbiology2026
To establish an indirect ELISA method for the detection of human brucellosis based on signal peptide-truncated recombinant Cu/Zn SOD and its preliminary clinical verification.
Article in Frontiers in cellular and infection microbiology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Background: Serological diagnosis of brucellosis is hindered by cross-reactivity with lipopolysaccharide (LPS) antigens, resulting in insufficient specificity and sensitivity, which remains one of the major bottlenecks in current clinical testing for brucellosis. Methods: To overcome this limitation, we developed an indirect ELISA based on recombinant Cu/Zn superoxide dismutase (rSOD) from Brucella. The N-terminal signal peptide of rSOD was truncated and expressed as a soluble His-tagged protein in Escherichia coli BL21 (DE3) through codon optimization. After purification, detection parameters were optimized using checkerboard titration, and the cutoff value was established using 40 sera from healthy blood donors (mean + 3SD). We evaluated the assay's performance against LPS-ELISA using 106 clinical samples (63 from brucellosis patients and 43 from healthy controls), assessing reproducibility, cross-reactivity with endemic infections (tuberculosis, rickettsiosis, and echinococcosis), and diagnostic accuracy. Results: Optimal conditions were determined as 1.25 μg/mL antigen concentration, serum dilution of 1:800, and a cutoff OD₄₅₀ of 0.386. Intra-batch and inter-batch coefficients of variation ranged from 0.93% to 4.60% and 1.09% to 12.88%, respectively. Cross-reactivity rates were 15% for tuberculosis, 5% for rickettsiosis, and 0% for echinococcosis. In clinical testing, the rSOD-ELISA demonstrated a sensitivity of 82.5% (52/63, 95% CI 71.4%-89.9%) and a specificity of 90.7% (39/43, 95% CI 78.4%-96.3%), with a false positive rate of 9.3%. Compared with LPS-ELISA, it showed comparable sensitivity (82.5% vs. 80.9%), higher specificity (90.7% vs. 83.7%), a lower false positive rate (9.3% vs. 16.3%), and an overall agreement of 96.2% (Kappa = 0.92, P < 0.001). Conclusion: The rSOD-ELISA demonstrates superior diagnostic performance through higher specificity and a lower false positive rate compared with conventional LPS-based tests. Although further validation through larger multicenter studies is needed, it represents a promising supplementary serological tool for brucellosis screening in areas with multiple concurrent pathogens.
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