Development of a cELISA based on a trivalent nanobody-HRP fusion protein targeting BP26 for brucellosis diagnosis

Brucellosis is a zoonotic disease that poses a serious threat to both humans and animals, not only creating a significant public health risk but also causing considerable economic losses. Timely and accurate diagnosis is critical for the effective prevention and control of brucellosis. However, current ELISAs target lipopolysaccharides (LPS) and often cross-react with other Gram-negative bacteria, resulting in limited specificity. Moreover, their high cost limits large-scale implementation and routine laboratory screening. In this study, a cELISA targeting the Brucella protein BP26 was developed using a trivalent nanobody-horseradish peroxidase fusion protein (3Nbs-HRP). The assay exhibited excellent performance, with a sensitivity of 99% and a specificity of 95.9%, and demonstrated high repeatability. High concordance was achieved compared with the serum agglutination test (SAT) and a commercial ELISA. In conclusion, these findings suggest that the assay is an efficient and cost-effective serological method suitable for clinical screening in routine diagnostic laboratory settings, with the potential to enhance brucellosis surveillance and control efforts.