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 was purified and antigenically characterized by use of monoclonal antibodies (mAbs). Purification was achieved from the sodium dodecyl sulphate-insoluble (SDS-I) cell wall (CW) fraction of vaccine strain B. abortus B19 which was shown by use of mAbs to contain the two major outer membrane proteins of 25 and 36 kDa linked to peptidoglycan, smooth lipopolysaccharide (S-LPS), and rough LPS (R-LPS). Purity of Omp25 was checked with a number of mAbs directed to the different components of the SDS-I fraction. In ELISA, five anti-Omp25 mAbs, which showed significant binding to B. abortus whole cells and which are probably directed to conformational epitopes well-exposed on the bacterial surface, reacted poorly or not at all with the purified Omp25. Addition of R-LPS to purified Omp25 restored the binding capacity of these mAbs, which suggested that R-LPS may play an important role in reconstitution and exposure of conformational epitopes of Omp25. Immunoelectron microscopy showed that Omp25 was inserted into the R-LPS vesicles. Four of these anti-Omp25 mAbs probably recognize the same or closely located epitopes on Omp25, since one of the mAbs conjugated to peroxidase was inhibited in its binding in ELISA by the three others. Other anti-Omp25 mAbs showed strong binding to purified denatured Omp25 and their binding capacity was not affected by the addition of R-LPS to the purified Omp25. Thus, these results confirmed, as defined by the mAbs, the presence of both sequential and at least one conformational epitope on Omp25.
Cloeckaert
A
A
Laboratoire de Pathologie Infectieuse et d'Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France.
Zygmunt
M S
MS
Bézard
G
G
Dubray
G
G
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D002003:Brucella abortus / Q000737:chemistry* / Q000276:immunology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000276:immunology*; D016253:Microscopy, Immunoelectron
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D008070:Lipopolysaccharides