project administration. Edited by Robert Haltiwanger == Supporting information == == References == == Associated Data == This section collects any data citations, data availability statements, or supplementary materials included in this article. == Supplementary Materials == == Data Availability Statement == All data are available in the main article, supporting figures/tables, and supporting Excel file (full microarray data and antibody sequences).. useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future. Keywords:antibody, antigen, carbohydrate, carbohydrate-binding protein, glycobiology, microarray, monoclonal antibody Abbreviations:ATCC, American Type Culture Collection; BHI, brain heart infusion broth; CDRH3, complementary determining region 3 of the heavy chain; CETE, carboxyethylthioethyl; DNP-BSA, dinitrophenylated bovine serum albumin; dPNAG, deacetylated PNAG; HC, heavy chain; HIC, hydrophobic conversation chromatography; HRP, horseradish peroxidase; IgG, immunoglobulin G; IgM, immunoglobulin M; KDO, ketodeoxyoctonic acid, 3-deoxy-d-manno-oct-2-ulosonic acid; LC, light chain; LLPC, long-lived plasma cell; mAb, monoclonal antibody; MW, molecular weight; PBST, PBS with Tween-20; PNAG, poly–1,6-N-acetylglucosamine; PSR, polyselectivity reagent; RT, room temperature; SEC, size-exclusion chromatography The immune system produces a diverse assortment of carbohydrate-binding antibodies that are vital for human health (1,2). For example, antibodies that bind bacterial, fungal, and other microbial polysaccharides provide protection from infections (3). These antibodies can be acquired through natural processes or can be induced through vaccination (4,5). In fact, Food and Drug Administrationlicensed carbohydrate-based vaccines targetingStreptococcus pneumoniae,Haemophilus influenzaetype b, andNeisseria meningitidisare administered routinely and have had a major impact on human health (6,7,8). Carbohydrate-binding antibodies also influence medical care in other ways (2). For instance, endogenous antibodies to ABO blood group antigens play a critical role in matching Adipor1 donors and recipients for blood transfusions (9). In addition to beneficial effects, antibodies that recognize self-glycans can contribute to autoimmune ZL0420 diseases, such as GuillainBarre syndrome (10,11). For these reasons, identifying and studying antibodies that target carbohydrates or glycans is ZL0420 crucial for a complete understanding of the immune system. While critically important, we know very little about individual antiglycan antibodies and their properties. From profiling serum antibodies on glycan microarrays, we know that many different glycans are recognized by endogenous antibodies (12,13,14,15). However, few details are available for the specific antibodies that bind to those glycans. For example, recognition of the various glycans could be due to: (1) a small number of polyreactive antibodies, ZL0420 (2) a mixture of many different highly selective antiglycan antibodies, or (3) some combination of various polyreactive and selective antibodies. In addition, it is not known if they are highly polyclonal or largely oligoclonal at the sequence level. We know very little about which V, D, and J genes are used to construct antiglycan antibodies, or how the immune system evolves and mutates those antibodies during the course of infection (16). Antiglycan antibodies are often thought to have ZL0420 broad specificity and low affinity, but these generalizations are based on very limited information (17,18,19). A key barrier to answering these questions is usually a lack of access to individual antiglycan antibodies, especially human antiglycan antibodies. Unlike antibodies to protein or peptide targets, generating monoclonal antibodies (mAbs) to many glycan targets remains an elusive challenge in the field (20). The dearth of antiglycan antibodies can be attributed to a combination of poor immunogenicity and the natural expression of glycans in healthy mammalian tissues, reducing the effectiveness of traditional hybridoma technologies. Alternative routes, such asin vitroselection platforms, have had some.