Paratuberculosis (Johne's disease) is a chronic enteritis that affects ruminants and is caused by Mycobacterium avium subsp. paratuberculosis. The disease is spread worldwide and causes important economic losses. In Brazil, M. paratuberculosis has been isolated, but there are no statistic studies about its prevalence. In this study, 200 sera selected from animals held at dairy farms in Rio de Janeiro State, Brazil, were tested for the presence of antibodies specific for M. paratuberculosis using an in-house ELISA test. This test employs a protoplasmic paratuberculosis antigen (PPA - Allied Monitor) as capture antigen and a monoclonal anti-bovine IgG conjugated to alkaline phosphatase (Sigma). Eighty-seven (43.5%) samples were reactive in the PPA-ELISA and 113 (56.5%) were negative. The results were compared to those obtained with the commercial ELISA test PARACHEK (CSL) and showed a sensitivity of 61.6% and a specificity of 70.2%, with 38.4% (33/86) of false negatives and 29.8% (34/114) of false positives. The use of the PPA-ELISA showed to be feasible and can be helpful for herd diagnosis to identify foci of Johne's disease.