B-017 Benchtop glyco-analyzer: from development of biomarker to potential use in clinical setting

Background Disease-related glycan alteration has attracted much attention as a promising biomarker candidate, especially in cancer or inflammation related diseases. Lectin is a naturally occurring functional protein that recognizes various glycan moieties and has a long history of use in glycan research. In the effort of omics-based biomarker development, lectin microarray (LMA), has accelerated the discovery of glyco-alteration and the candidate verification with high sensitivity in detection and simplicity in sample pretreatment. However, large-scale validation is still challenging because of the manual operation process and chip lot-to-lot variation in LMA. Methods An automated analytical system, GlycoBIST1-3, a milli-sized lectin bead array handled by simple robotics, was used. To overcome the above issue, we improved the simplicity and throughput by shortening all reaction processes to be completed around 30 min and adopting a touch panel interface. For an entry of analysis, a “standard tip” comprising 15 lectins with different binding specificities was designed, which can be produced in a thousand scale of the same lot. The utility was evaluated using 12 polyacrylamide (PAA)-glycan conjugates. The reliability was confirmed by repeatability tests and long-term storage tests. A lectin/antibody hybrid bead array using the antibody against a target biomarker was adopted to detect glyco-alteration more precisely. Results Twelve different PAA-glycan conjugated with mono/di-/trisaccharides clearly highlighted distinct binding specificities of 15 lectins in the standard tip. In a within-run reproducibility test, the measurement results indicated that the maximum CV value was 13.5%, and the average CV for the 15 lectins was 8.2%. A day-to-day reproducibility test showed that most lectins had a CV of less than 10% during 5-days. For the first multilectin/antibody beads array application, serum M2BP, whose glycosylation isomer has been used as a liver fibrosis marker approved by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan 4,5, was verified. M2BP was preliminarily immunoprecipitated from HCC sera and then subjected to the glycan profiling with an automatic 32-min operation for the hybrid beads array. The preliminary data was consistent with that of the lectin microarray, suggesting the possibility of M2BP subtyping. Conclusions A fully automated glyco-analyzer has realized rapid glycan analysis in around 30 minutes with sufficient accuracy and versatility. With the ability to detect glycan qualitative change on a target protein, it would help to detect disease-related glycan alteration more precisely, leading to the development of an effective glyco-biomarker. This rapid and user-friendly system would accelerate glyco-biomarker development, including the validation phase, and could be used in clinical settings in the future. Reference: 1) Shimazaki, H., et al. Anal. Chem. 2019, 91; 11162-11169, 2) Shimazaki, H., et al. Curr. Protoc. Protein Sci. 2020, 99; e103, 3) Fuseya, S., et al. J. Visualized Exp. In press, 4) Kuno, A., et al. Sci. Rep. 2013, 3, 1065. 5) Yamasaki, K., et al. Hepatology 2014, 60, 5,1563-1570