Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-
polysaccharide (OPS). Serology is the most cost-effective means of detecting
brucellosis, as
infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyl residues (D-Rha4NFo) that are variably α(1→2)- and α(1→3)-linked. Six d-Rha4NFo homo-
oligosaccharides were synthesized, each containing a single α(1→3) link but with a varied number of α(1→2) links. After conjugation to
bovine serum albumin (BSA),
glycoconjugates 1 to 6 were used to develop individual indirect
enzyme-linked
immunosorbent assays (iELISAs). The diagnostic capabilities of these
antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the
complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth
lipopolysaccharide (sLPS) iELISA, and competitive
enzyme-linked
immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the
synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1→3)-linked
disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic
trisaccharide antigens 2 and 3 and tetrasaccharide
antigen 4 identified an OPS
epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic
oligosaccharide conjugates function as effective surrogates for naturally derived
antigens. The creation of discrete OPS
epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds significance for the design of
brucellosis vaccines and diagnostics that enable the differentiation of infected from vaccinated animals.