PURIFICATION AND ANTIGENIC ANALYSIS OF THE MAJOR 25-KILODALTON OUTER-MEMBRANE PROTEIN OF BRUCELLA-ABORTUS

The major 25-kDa outer membrane protein (Omp25) of Brucella abortus wa s purified and antigenically characterized by use of monoclonal antibo dies (mAbs). Purification was achieved from the sodium dodecyl sulphat e-insoluble (SDS-I) cell wall (CW) fraction of vaccine strain B. abort us B19 which was shown by use of mAbs to contain the two major outer m embrane proteins of 25 and 36 kDa linked to peptidoglycan, smooth lipo polysaccharide (S-LPS), and rough LPS (R-LPS). Purity of Omp25 was che cked with a number of mAbs directed to the different components of the SDS-I fraction. In ELISA, five anti-Omp25 mAbs, which showed signific ant binding to B. abortus whole cells and which are probably directed to conformational epitopes well-exposed on the bacterial surface, reac ted poorly or not at all with the purified Omp25. Addition of R-LPS to purified Omp25 restored the binding capacity of these mAbs, which sug gested that R-LPS may play an important role in reconstitution and exp osure of conformational epitopes of Omp25. Immunoelectron microscopy s howed that Omp25 was inserted into the R-LPS vesicles. Four of these a nti-Omp25 mAbs probably recognize the same or closely located epitopes on Omp25, since one of the mAbs conjugated to peroxidase was inhibite d in its binding in ELISA by the three others. Other anti-Omp25 mAbs s howed strong binding to purified denatured Omp25 and their binding cap acity was not affected by the addition of R-LPS to the purified Omp25. Thus, these results confirmed, as defined by the mAbs, the presence o f both sequential and at least one conformational epitope on Omp25.