Hybridoma cell lines which produce and secrete monoclonal antibodies which selectively bind to Mycobacterium avium subspecies paratuberculosis have been produced. Cells of M. avium subspecies paratuberculosis in biological samples may be detected and quantified by contacting the sample with the antibodies to form a M. avium subspecies paratuberculosis/antibody immunocomplex when M. avium subspecies paratuberculosis is present, which immunocomplex may then be detected. The monoclonal antibodies also may be incorporated into kits for the detection and quantification of M. avium subspecies paratuberculosis.