Johne's disease (JD) is a chronic intestinal infection of ruminant livestock caused by infection with Mycobacterium avium subspecies paratuberculosis. JD is an economically significant disease that is widespread and worldwide. Current control measures are either diagnosis/management or vaccination. There is a need for better tools and reagents to effectively control the disease. A number of antigens within the protein/peptide complement of Johnin purified protein derivative (PPD) and cell free culture supernatants of M. avium subspecies paratuberculosis are capable of eliciting a strong interferon-g release response early in the course of disease. The aim of this project is to identify and characterise a variety of antigens derived from culture media suitable for improved diagnosis by cell mediated immunity assay (IFNg release assay, BOVIGAM^TM, CSL Animal Health Ltd). A strategy involving a liquid phase proteomics approach was employed for rapid identification of suitable secreted proteins of M. avium and M. avium subspecies paratuberculosis. This consisted of preparative isoelectric focusing separation followed by SDS-PAGE and whole gel protein elution. Liquid phase separation allowed continuous monitoring of IFNg release assay activity of fractionated protein samples. 15 proteins were identified in active fractions applying direct N-terminal sequencing. Subsequently genes encoding these identified antigens were expressed in various systems and the recombinant proteins tested for their suitability in the diagnostic test. This approach is being utilized in conjunction with recombinant antigen screening.