Antigenic phenol-phase soluble lipopolysaccharide isolated from Brucella abortus 1119-3 by hot phenol-water extraction was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, controlled hydrolysis, periodate oxidation, methylation, and 'H and 1 C nuclear magnetic resonance studies to be an S-type lipopolysaccharide which could be cleaved to yield a lipid A and an 0-chain polysaccharide identified as an unbranched linear homopolymer of 1,2-linked 4,6-dideoxy-4-formamido-ft-D-mannopyranosyl residues. The serological reactivity of bovine antiserum to B. abortus 1119-3 with the lipopolysaccharides of Yersinia enterocolitica serotype 0:9 and Vibrio cholerae species has now been related to the occurrence of 1,2-linked Nacylated 4-amino-4,6-dideoxy-a-D-mannopyranosyl units in the 0-chain polysaccharides of their lipopolysaccharides.The diagnosis of brucellosis in humans and animals is frequently difficult to establish. Often the infection either is subclinical or yields varied responses in the host. Diagnosis, therefore, has frequently been based on the detection and quantification of Brucella antibodies in serum samples by agglutination, precipitin, complement fixation, and other methods (11). More recently, enzyme immunoassay tests with crude or purified antigens have been introduced (4, 20-22). False-negative and nonspecific reactions may be problems in these test systems. This report describes our work on the isolation, purification, and determination of the structure of a unique Brucella surface antigen which could be used both for diagnostic tests and for studies involving the production and use of monoclonal antibodies for the identification of Brucella abortus.The present investigation led to the identification of the antigenic 0-chain polysaccharide of B. abortus S-type lipopolysaccharide (LPS) as a linear homopolymer of 1,2-linked 4,6-dideoxy-4-formamido-a-D-mannopyranosyl units.(This paper was presented previously

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and 0 polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica 0:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype Aand M-antigen structures, an interpretation supported by competitive binding studies with 0 polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous al,2-linked 4,6-dideoxy-4-formamido-Dmannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by al,2-linked trior tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element ft-D-Rha4NFo(l-*3)a-D-Rha4NFo bracketed by al,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated al,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.

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Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS‐3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS‐3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti‐BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme‐linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.