Polyclonal and monoclonal antibodies against mycobacteria are frequently reactive with heat shock protein (hs60, Ubiquitin,..). These proteins present in all organism, have been implicated in autoimmune disease. Here we describe a monoclonal antibody (MF.D4B6) that recognizes an epitope on a 25/45 kDa antigen on Mycobacterium paratuberculosis. The reactivity of MF.D4B6 antibody was studied by Western-blot on different bacterial species such as Mycobacterium phlei, Escherichia coli, Bacillus subtilis and PPA-3, a commercial antigen from Mycobacterium avium. In all of them, the epitope on the 25/45 kDa was detected as well as additional antigens. Furthermore membrane leukocyte antigens from goats and sheep (45 kDa), cattle (65 kDa), swine (52 and 65 kDa) and humans (30 and 60 KDa), were detected by MF.D4B6. Cross-reactive monoclonal antibodies such as MF.D4D6 are useful for studying cross-reactive epitope in the autoimmune process related to mycobacterial disease.