Introduction

Bovine paratuberculosis is caused by infection of young calves with Mycobacterium avium ssp. paratuberculosis (MAP), and results in chronic granulomatous infection of the ileum. Relatively little is known about immunogenic properties of individual antigens of MAP. 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.

Materials and methods

Mouse monoclonal antibodies were generated against recombinant MAP 70 kD heat shock protein (Hsp70) using conventional hybridoma technology. Antibodies produced by different hybridomas were primarily screened using Hsp70 as antigen in ELISA. 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.

Results

In total, 8 hybridomas which recognized MAP Hsp70 were generated. Five hybridomas produced antibodies recognizing conformational epitopes on the Hsp70. Three of these hybridomas produced monoclonal antibodies recognizing 2 different linear epitopes of Hsp70. The 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.

Conclusion

This study described the presence of multiple B cell epitopes in MAP Hsp70. Monoclonal antibodies recognizing linear epitopes of MAP Hsp70 were generated, which may prove useful in a number of commonly used techniques to study the presence of MAP in tissue and various substrates.