Characterization of polysaccharide conformational epitopes by surface plasmon resonance

SPR techniques can provide a wealth of insight into the nature of protein-carbohydrate interactions. Information not obtained readily by other methodologies can be gathered relatively quickly in a label-free manner with low sample consumption. This chapter focused on two applications in which SPR has been used to map conformational epitopes on bacterial polysaccharides recognized by protective antibodies. In one example, methods for demonstrating the conformational nature of the epitope recognized by an anti-GBS antibody were described. Dramatic epitopic stabilization at 2 repeating units with further significant stabilization between 7 and 20 repeating units was demonstrated. In a second example, SPR methods were employed in characterization of the epitope recognized by a protective antibody against the group B meningococcus. It was shown that the antibody bound a long epitope, in excess of eight monosaccharides and probably helical, on NPr-GBMP but did not bind to GBMP. The binding of the protective antibody to GBMP only when GBMP is cell associated, or with an attached lipid, indicated that the protective GBM epitope consists of more than GBMP. NPr-GBMP mimics a cell surface complex consisting of extended helical portions of the GBMP in association with a second molecule, possibly a phosphoglycerolipid. SPR experiments indicated that the protective nature of certain antibodies induced by the NPr-GBMP vaccine is attributable to their recognition of an abundant internal epitope on NPr-GBMP and cell-associated GBMP. A nonprotective antibody, specific for NPr-GBMP, recognized a terminal and consequently minor epitope on the polysaccharide. Low levels of this nonprotective antibody binding to cells and unpurified polysaccharide confirmed recognition of a minor epitope on the natural antigen as well. In contrast, a protective antibody exhibited a high level of binding to the cell-associated antigen.