Technical Abstract: As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial ELISA that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include: specific antibody responses were detected ~90 -100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection / sensitization with non-tuberculous Mycobacteria spp., and the apparent sensitivity / specificity of the ELISA with naturally infected cattle was 63% / 98 % respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.