Application of Brucella type IV secretion proteins VirB1, VirB5, and VirB6 in the serological diagnosis of brucellosis

Brucellosis, a significant zoonotic infectious disease caused by Brucella spp., necessitates the development of efficient, rapid, accurate, and cost-effective diagnostic methods. This study evaluated the diagnostic value of Brucella Type IV secretion proteins VirB1, VirB5, and VirB6 for human brucellosis serological diagnosis. A total of 100 positive serum samples, 96 negative serum samples, and 27 serum samples from patients with fever caused by other pathogens were collected. Recombination VirB1 (rVirB1), rVirB5, and rVirB6 were expressed using prokaryotic expression systems, while VirB2 and VirB7 were synthesized as peptides. An indirect ELISA method was established using purified proteins and synthetic peptides, with sensitivity, specificity, AUC, and cut-off values determined through ROC analysis. The study successfully prepared rVirB1 (~ 36 kD, purity 90.6%), rVirB5 (~ 32 kD, purity 90.3%), and rVirB6 (~ 28 kD, purity 94.9%). These proteins exhibited sensitivity and specificity exceeding 0.9500 and 0.9271, respectively, comparable to traditional LPS antigens. Cross-reactivity assessments revealed that rVirB1 exhibited cross-reactivity with Pseudomonas putrida, while rVirB5 and rVirB6 showed no cross-reactivity. In contrast, LPS exhibited 14 instances of cross-reactivity. This study successfully developed an indirect ELISA diagnostic method using rVirB1, rVirB5, and rVirB6, demonstrating high diagnostic accuracy comparable to traditional LPS antigen detection techniques. Although some cross-reactivity was observed, the method presents a promising new candidate for the serological diagnosis of brucellosis. Future research should focus on optimizing this method to enhance diagnostic specificity and reliability.