Bovine paratuberculosis is caused by infection of young calves with Mycobacterium avium ssp. paratuberculosis (MAP), and results in chronic granulomatous infection of the ileum. A recent vaccination-challenge study (Koets et al. Vaccine 2006) identified the recombinant 70 kD heat shock protein (Hsp70) of MAP as a promising subunit vaccine candidate. Contrary to expectations the major post vaccination immune response associated with protection appeared to be an antibody response. The aim of the present study was to define antibody epitopes of the 70 kD heat shock protein of MAP through the generation of monoclonal antibodies.

Mouse monoclonal antibodies were generated against recombinant MAP 70 kD heat shock protein (Hsp70) using conventional hybridoma technology. Subsequent epitope mapping was performed using synthetic peptides representing different parts of the Hsp70. Monoclonal antibodies recognizing linear epitopes were tested for use in western blot, immunohistochemistry and electronmicroscopy. Sera from cattle and goats infected with MAP and/or vaccinated with Hsp70 were used to determine if linear epitopes were recognized.

In total, 8 hybridomas which recognized MAP Hsp70 were generated. Five hybridomas produced antibodies recognizing conformational epitopes on the Hsp70. Three hybridomas produced monoclonal antibodies recognizing 2 different linear epitopes of Hsp70. These 3 antibodies could successfully be used in peptide specific ELISA, western blots, immunohistochemistry and electronmicroscopy of tissues of animals infected with paratuberculosis. One epitope was conserved in multiple mycobacterial species, with the other epitope differentiation between MAP and M. tuberculosis / M. bovis was possible, however this epitope appeared to be present in at least some of the M. avium ssp. avium species tested. The conserved N-term linear epitope was also recognized by cattle and goats vaccinated with Hsp70. In the less conserved C-term of the protein different epitopes were recognized by different species.

In conclusion, monoclonal antibodies recognizing linear epitopes of MAP Hsp70 were generated, which may prove useful in commonly used techniques to study the presence of MAP in tissue and various substrates. This study also showed that antibodies are induced against multiple linear B cell epitopes of MAP Hsp70 following vaccination of cattle and goat with Hsp70.