A multiplex suspension array for screening of carbohydrate binding proteins and influenza

Carbohydrate-protein interactions are involved in many crucial biological processes, from protein stability, to immune response, to pathogen infection. A fast and reliable method to determine carbohydrate binding affinity and specificity was developed as an alternative to ELISA and glycan microarray screening.Optically distinct neutravidin-coated microspheres were functionalized with biotinylated carbohydrates, to create a glycan array in suspension format. A range of fluorescently labeled carbohydrate binding agents including lectins, toxins, and viruses were incubated with the glycan suspension array. In addition to characterizing carbohydrate binding preference, simultaneous strain typing of influenza virus was performed using additional multiplex microspheres conjugated to antibodies specific for influenza A subtypes. Inactivated virus was incubated with the microspheres, detected using fluorescence-labeled anti-influenza virus antibodies, and measured by flow cytometry.Biotinylated carbohydrates were successfully captured by neutravidin conjugated microspheres as observed by specific binding of fluorescently labeled lectins SNA-I and MAA and Cholera toxin B (CTB) subunit. Antibodies were also conjugated to microspheres allowing for multiplex analysis of influenza virus sub-type and receptor specificity of human influenza A subtype H1N1 and avian influenza A subtype H5N1. Each subtype of influenza virus bound only to those microspheres displaying carbohydrates with its specific receptor and anti-influenza A antibody. These results have been confirmed previously by other methods, including hemagglutination and glycan microarray.This approach provides improved control over carbohydrate surface density, increased throughput for the analysis of many samples and conditions, reduced sample volumes, and better quantification.