Serological assays for
bovine tuberculosis diagnosis require the use of multiple Mycobacterium bovis specific
antigens to ensure the detection of infected animals. In the present study, identification and selection process of
antigens, based on data from published proteomic studies and involving the use of bioinformatics tools and an immuno-screening step, was firstly performed for identifying novel
antigens that elicit an antibody response in M. bovis
infection. Based on this approach, a panel of 10 M. bovis
antigens [with known relevance (MPB70, MPB83, MPB70/83, and ESAT6/CFP10) and novel (Mb1961c, Mb1301c, Mb3871, Mb1403, Mb0592, and PE25/PPE41)] were constructed and thenused to develop a new multiplexed serological assay based on Luminex technology. The performance of the Luminex-bTB immunoassay was evaluated using sera from cattle with known
tuberculosis status. Among the
proteins whose ability to detect
bovine tuberculosis was evaluated for the first time, PE25/PPE41 and Mb1403, but not Mb3871, showed good detection capacity. Following multiple
antigen combination, the final Luminex-bTB immunoassay included seven
antigens (MPB70, MPB83, MPB70/83, ESAT6/CFP10, PE25/PPE41, Mb1403, and Mb0592) and showed better global performance than the immunoassay using the four usual
antigens (MPB70, MPB70/83, MPB83 and ESAT6/CFP10). The specificity and sensitivity values were, respectively, of 97.6% and 42.8% when the cut-off of two-positive
antigens was used to classify samples as positive. With the use of the more-restrictive criterion of three-positive
antigens, the specificity increased to 99.2% but the sensitivity decreased to 23.9%. The analysis of
antigen profiles generated with the Luminex-bTB immunoassay showed that mainly serodominant
proteins were recognized in samples from infected cattle. The detection of Mb1961c and Mb1301c appeared to be associated with presumed false-positive results. Moreover, sera from cattle originating from bTB-outbreaks but having inconclusive or negative skin test results were identified as positive by the Luminex-bTB immunoassay and showed an
antigen pattern associated with M. bovis
infection. The Luminex-bTB immunoassay including seven
antigens may be useful as adjunct test for the detection of M. bovis-infected herds, and different cut-offs could be applied according to the
bovine tuberculosis epidemiological context.