Production of Recombinant Antigens for Use in the Serological Diagnosis of Animal Brucellosis

Background: Brucellosis, a global zoonotic disease, poses significant threats to both public health and economic stability, particularly in livestock industries, necessitating the development of improved diagnostic methods for early detection and control. The development of recombinant antigens offers a promising approach to improve diagnostic accuracy and standardization of test systems.Methods: Genes encoding Brucella antigens (Omp2b, BP26, and SodC) were cloned into the pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). Recombinant proteins were purified using Ni-NTA chromatography and characterized for expression level, solubility, yield, and stability. Diagnostic performance was evaluated using the same complete bovine serum panel of 86 samples, comprising 56 serologically positive and 30 serologically negative sera. Each of the 86 serum samples was tested with all three individual antigens (Omp2b, BP26, and SodC) and all three combined-antigen formulations (BP26+Omp2b, BP26+SodC, and BP26+Omp2b+SodC); no separate sub-panels were used. Sensitivity and specificity were therefore calculated for every antigen format using the same reference panel, followed by comparisons with commercial ELISA kits. Paired differences in sensitivity and specificity were evaluated using Cochran’s Q test followed by Holm-adjusted pairwise McNemar tests.Results: Successful cloning and expression yielded proteins of expected molecular weights (38, 26, and 18 kDa). BP26 demonstrated the highest expression (28.4%), solubility (82.1%), and yield (14.8 mg/L), along with superior stability and reproducibility. The verified sensitivity/specificity values were 80.6%/88% for Omp2b, 91.7%/95% for BP26, 75%/90% for SodC, 94.4%/92% for BP26+Omp2b, 95.8%/93% for BP26+SodC, and 97.2%/91% for BP26+Omp2b+SodC. The new tests also showed improved stability, reduced cross-reactivity, and better detection at low antibody titre compared to commercial kits.Conclusion: Recombinant antigens, particularly BP26 and its combinations, show improved diagnostic performance in the serological diagnosis of animal brucellosis. The developed antigen systems demonstrate high sensitivity, specificity, and stability, supporting their potential application in advanced diagnostic test development.KeywordsBrucellosis, Recombinant antigens, BP26, Omp2b, SodC, Serological diagnosis, ELISA, Diagnostic sensitivity