The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of the smooth lipopolysaccharide. Infection by various Brucella biovars causes abortions and infertility in a wide range of domestic and wild animals and debilitating disease in humans. Diagnosis relies on the detection of antibodies to the A and M antigens expressed in the O-polysaccharide. This molecule is a homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-d-mannopyranose (Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides only available by chemical synthesis and conjugated via reducing and non-reducing residues to bovine serum albumin have revealed the structural basis of the fine specificity that allows the discrimination of these closely related A and M epitopes. All three M specific monoclonal antibodies (mAbs) are inferred to possess groove type binding sites open at each end, and recognize an α1,3 linked Rha4NFo disaccharide as a part of a trisaccharide epitope, which in two mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is sufficiently large to engage up to six Rha4NFo residues and involves weak recognition of α1,2 linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M tetrasaccharide. Two A specific mAbs also possess groove type binding sites that accommodate six and four α1,2 linked Rha4NFo residues.