Development and evaluation of an ELISA based on recombinant Mb1454 antigen for the detection of specific anti-Mycobacterium bovis antibodies in cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a zoonosis of global concern due to the absence of effective vaccines or treatments. The conventional diagnostic method, the single intradermal comparative cervical tuberculin test (SICTT), is limited by cost and sensitivity, hindering control strategies. To overcome these limitations, an ELISA that detects specific IgG antibodies against M. bovis using the recombinant protein Mb1454 was developed. The optimized ELISA conditions included 0.5&#x202f;&#xb5;g of recombinant Mb1454 protein for plate sensitization, PBMT blocking buffer (PBS + 1&#x202f;% BSA + 3&#x202f;% milk + 0.05&#x202f;% Tween-20), serum dilution of 1:200, secondary antibody dilution of 1:1000, 1&#x202f;h incubation, and 15&#x202f;min substrate development time. The analysis of the ELISA-Mb1454 in M. bovis-positive (bTB+, n&#x202f;=&#x202f;113) and negative (bTB-, n&#x202f;=&#x202f;120) bovine serum samples showed 92.9&#x202f;% sensitivity and 90.0&#x202f;% specificity, with an area under the ROC curve of 0.96, indicating high discrimination capacity in M. bovis detection (p&#x202f;<&#x202f;0.0001). The assay demonstrated high analytical specificity, with no cross-reactivity observed in sera from animals infected with other common pathogens, including BoAHV-1, BoAHV-5, BVDV, Neospora caninum, Brucella abortus, and Leptospira sp. The results showed no correlation between circulating IgG antibody levels against the Mb1454 antigen and the pathological stage of bovine tuberculosis (bTB) lesions. Nonetheless, our findings demonstrated the immunogenicity of the Mb1454 protein and discriminatory performance of the ELISA-Mb1454 assay in differentiating bTB-positive and -negative cattle, even in the absence of a direct association between antibody titers and disease severity.