Summary This application addresses a major unmet need for technology that will facilitate the integration of glycomics and glycan expression with genomics, transcriptomics, and metabolomics. The rapidly developing field of glycoscience must have tools to identify and characterize the expression of human and animal glycans in the context of normal development, homeostasis, and in disease processes. Traditional studies in the glycosciences have relied on an odd assortment of plant-derived lectins and a few traditional mouse monoclonal antibodies, but such reagents have multiple limitations. This application exploits our breakthrough technology that allows us to develop suites of specific, rigorously characterized anti-carbohydrate antibodies (ACAs). We have developed high-throughput immunization and screening technologies using variable-lymphocyte receptors (VLRBs) that are generated upon immunization of ancient sea lampreys with cells, cell-conjugates, glycoproteins, or tissues. Because of the evolutionary diversity of both their immune systems and glycomes as compared to mammals, these ancient fish generate a diverse repertoire of anti-carbohydrate VLRBs with exquisite specificity, and are able to discriminate between different linkages, motif presentation on N- and O-glycans, and the presence of functional groups on individual monosaccharides. After immunization, the cloned cDNAs encoding the VLRBs are expressed in a yeast surface display (YSD) library containing multiple individual specificities. This innovative technology development allows us to both generate a ‘library of VLRBs’ in a single immunization protocol, as well as sequentially screen these stable YSD libraries for ACAs using our numerous and diverse glycan and glycopeptide microarrays and display technologies. Ultimately, after the VLRBs are screened for their binding to glycan antigens, recombinant and reproducible VLRB-Ig chimeras are expressed, which can be used for all types of immunological approaches to explore glycan expression and function. We propose 3 robust and innovative Specific Aims to address the current technological deficiencies: Aim 1- Refine and expand our technology to generate permanent VLRB YSD libraries against a diverse array of human glycan targets, using multiple immunization and screening strategies. Aim 2- Enrich for and characterize monoclonal VLRBs that are specific for human glycans, which will be critical in mapping the Human Glycome, by mining the created YSD libraries. Aim 3- Generate novel VLRB specificities using targeted mutagenesis of characterized VLRBs, to create tailored antibody specificities with potentially higher affinities. The success of our studies will climax with an arsenal of rigorously vetted tools and robust reagents that will be made publicly available and accessible to the broader research community. This will ultimately encourage widespread study of these incredibly important, complex molecules that are vital in human health a...