Multidimensional glycan arrays for enhanced antibody profiling.

Carbohydrate-binding antibodies play a critical role in basic and clinical research. Monoclonal antibodies that bind glycans are used to measure carbohydrate expression, and serum antibodies to glycans can be important elements of the immune response to pathogens and vaccines. Carbohydrate antigen arrays, or glycan arrays, have emerged as powerful tools for the high-throughput analysis of carbohydrate-protein interactions. Our group has focused on the development and application of neoglycoprotein arrays, a unique array format wherein carbohydrates are covalently attached to a carrier protein prior to immobilization on the surface. The neoglycoprotein format permits variations of glycan structure, glycan density, and neoglycoprotein density on a single array. The focus of this study was on the effects of neoglycoprotein density on antibody binding. First, we evaluated binding of five monoclonal antibodies (81FR2.2, HE-195, HE-193, B480, and Z2A) to the blood group A antigen and found that neoglycoprotein density had a substantial effect on recognition. Next, we profiled serum antibodies in 15 healthy individuals and showed that inclusion of multiple neoglycoprotein densities helps distinguish different subpopulations of antibodies. Finally, we evaluated immune responses induced by a prostate cancer vaccine and showed that variations in neoglycoprotein density enable one to detect antibody responses that could not be detected otherwise. Neoglycoprotein density is a useful element of diversity for evaluating antibody recognition and, when combined with variations in glycan structure and glycan density, provides multidimensional glycan arrays with enhanced performance for monoclonal antibody development, biomarker discovery, and vaccine optimization.