Luminex multiplex glycan array (LMGA): a high throughput and high content platform for GBPs

Glycans play an essential role in the maintenance of diverse cellular functions such as cell adhesion, signal transduction and immune response through interaction with a large number of glycan-binding proteins (GBPs) such as C-type lectins, galectins, siglecs and microbial GBPs. Another main category of GBPs is antiglycan antibodies that are critical determinants for blood transfusion and may also have a tremendous potential as diagnostic and prognostic markers for many diseases including cancer and autoimmunity. Despite the critical importance of GBPs, progress in this field has been slow due to the lack of powerful technologies for evaluation of glycan structures recognized by GBPs. This obstacle was partially alleviated by advent of the solid surface glycan arrays, which greatly enhanced our understanding of glycan functions. However, solid glycan array is technically challenging and cannot be easily made available to non-experts and it is not a suitable clinical tool. With a recent R21 award, we have developed the first generation of Luminex Multiplex Glycan Array (LMGA) containing 202 glycans. LMGA is a technology that meets all the requirements for large scale GBP analyses. Our pilot studies with LMGA-202 demonstrated the feasibility of using LMGA to simultaneously analyze many mammalian GBPs, lectins, recombinant anti-glycan antibodies, and natural antiglycan antibodies in serum/plasma. This platform is capable of analyzing up to 500 glycans in a single assay for 1020 samples per day per technician. We have successfully demonstrated that LMGA has very high sensitivity and specificity, is cost-effective and flexible in glycan composition, has a rapid turn-around time, and requires small sample volume. These attributes render the platform accessible by all biologists and an excellent choice for clinical testing. The first major goal of this U01 is to create the second generation of LMGA that will comprise at least 1000 glycans covering the diverse glycospace (LMGA-1K). The second major goal is to make LMGA less complex and more affordable via streamlining the procedures and to make the technology accessible to all biologists with interest in glycobiology through four complimentary approaches: a) provide core services for custom samples including lectins, GBPs, antiglycan antibodies, natural antiglycan antibodies in serum/plasma samples as well as cells and microbes using our high throughput and high content LMGA infrastructure; b) conjugation and testing of custom glycans against their GBPs; c) provide our ready-to-use glycan beads for others to use in their own laboratories; and d) develop and provide a standard operation protocol (SOP), QC reagents, bioinformatics tools, training and technical assistance, and a quality evaluation program so that other users can establish the LMGA platform in their own laboratories.