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Volume 138, Issue 7

Demonstration of peptidoglycan-associated outer-membrane proteins by use of monoclonal antibodies Free

Abstract

A monoclonal antibody (3D6) was produced which reacted only with sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated and sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B115 than with that of . This mAb was also used in immunogold electron microscopy with whole cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs. Therefore we propose that variation in the apparent molecular mass of the major OMPs (25-27, 31-34, and 36-38 kDa) is due to varying numbers of PG subunit residues rather than attached R-LPS or S-LPS molecules. Our results suggest a very strong, possibly covalent, interaction of the major OMPs (25-27, 31-34 and 36-38 kDa) with PG. There is no evidence of association of the minor OMPs (10, 16.5, 19 and 89 kDa) with PG, since mAbs specific for these proteins reacted with single bands that were not apparent when reacted with the anti-PG mAb. Furthermore, lysozyme treatment did not affect the electrophoretic mobility of these proteins.

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© Society for General Microbiology, 1992
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1992-07-01
2023-06-05
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Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies
Microbiology 138, 1543 (1992); https://doi.org/10.1099/00221287-138-7-1543
/content/journal/micro/10.1099/00221287-138-7-1543
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