Monoclonal antibodies (MAbs) were produced to cytosoluble proteins (CP) with apparent molecular masses of 12, 24 and 28 kDa (CP12, CP24 and CP28) which were previously shown by immunoblotting to differentiate antibody responses of infected sheep from those of strain Rev. 1 vaccinated sheep. These MAbs were derived from mice infected with virulent smooth (S) strain H38. Most MAbs obtained were directed to CP28, which indicated (as was shown in infected sheep) that this protein was also highly immunogenic in mice. A large number of MAbs that showed reactivity to CP in ELISA but did not show reactivity in immunoblotting of CP were also obtained and might recognise conformational epitopes of these proteins. MAbs were used to localise CP12, CP24 and CP28. None of the MAbs reacted with whole cells in ELISA but showed reactivity with sonicated bacteria in ELISA, which indicated an internal localisation of these proteins. Among several B115 subcellular fractions tested, the anti-CP12 and anti-CP28 MAbs reacted essentially with the CP extract (CPE) in both ELISA and immunoblotting, whereas the anti-CP24 MAbs reacted with both CPE and cell envelope fraction (CEF) - although with lower intensity to the latter fraction. The internal localisation of these proteins was confirmed by immuno-electron microscopy of thin-sectioned B115 or 16M cells. Immunogold labelling was mainly observed in the cytoplasm and, consequently, CP12, CP24 and CP28 are probably cytoplasmic proteins. Immunoblotting of whole cell lysates with the MAbs also showed the presence of these proteins in all species and biovars, including the vaccine strains Rev. 1 and B19. The use of these MAbs should help further study of antibody responses in sheep and other hosts and may be of considerable value for developing new diagnostic tests for ovine brucellosis.


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