Technical Abstract: Antibody responses are useful indicators of Mycobacterium bovis infection of cattle. Tests for serological responses often use panels of multiple M. bovis antigens as detection probes. This is recommended because responses to single antigens may be too variable for consistent diagnosis. However, the use of multiple antigens increases costs and the risk of cross reactivity with antibodies directed against orthologous antigens expressed by other species. An alternative approach is to use a single antigen in a detection system with high analytical sensitivity. This approach is used in the SeraLyte-TBTM system, a sensitive chemiluminescent test for antibodies that bind to ferrite particles coated with the MPB83 antigen. SeraLyte testing was conducted in blinded fashion on frozen sera (N = 90 samples) collected from experimentally infected and control cattle (N = 71 animals). The sample included sera from animals infected with M. bovis (N = 27), M. kansasii (N = 4), M. avium subspecies paratuberculosis (N = 11), M. avium subspecies avium (N = 12) and uninfected animals (N = 17). As with all serological tests that utilize MPB83, reactivity was expected in M. bovis and M. kansasii-infected animals, but not in M-avium-infected and uninfected animals. Upon unblinding, sensitivity relative to known infection was 90% among the 71 individual animals, and 92% among the 90 individual samples tested. Specificity was 100%. The results indicate that SeraLyte-TB(TM) has sufficient sensitivity for detecting infection based on serological response to a single antigen.