Development and Preliminary Evaluation of an OMP16-Targeting Trivalent Nanobody-HRP-Based cELISA for Serological Detection of Bovine Brucellosis

Brucellosis is a globally prevalent zoonotic disease that imposes considerable economic burdens on the livestock industry and remains a significant threat to public health. Although lipopolysaccharide (LPS)-based serological assays are widely used in routine diagnosis, their inherent limitations—particularly cross-reactivity with other Gram-negative bacteria—underscore the need for the development of diagnostic approaches based on non-LPS antigens. In this study, we developed a trivalent nanobody–horseradish peroxidase (3Nbs-HRP) fusion protein targeting Brucella OMP16 and established a cELISA for the serological detection of bovine brucellosis. The diagnostic performance of the assay was assessed using 204 Brucella antibody-negative and 123 Brucella antibody-positive bovine serum samples. ROC curve analysis yielded a sensitivity of 87.7% and a specificity of 89.4%, with no significant cross-reactivity observed. By employing a recombinant antigen and a 3Nbs-HRP probe, this assay enhances biosafety and demonstrates strong potential for standardization and large-scale application, serving as a complementary tool to conventional LPS-based assays for the surveillance, diagnosis, and control of bovine brucellosis.