Reddy Revathi, Carpenter Eric, Halpin Anne, Sojitra Mirat, Peng Chuanhao, Lima Guilherme Meira, Xue Xiaochao, Yan Kejia, Pearcy Jean, Ellis Maria, Motyka Bruce, Lowary Todd L, West Lori, Derda Ratmir
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Alberta Transplant Institute and Canadian Donation and Transplantation Research Program, University of Alberta, Edmonton, AB, Canada.
Glycobiology. 2025 Aug 12. doi: 10.1093/glycob/cwaf042.
We test the performance of the multiplexed liquid glycan array (LiGA) technology in serological assays. Specifically, we use LiGA to detect ABO blood group antibodies in human serum. This LiGA, which we name ABO-LiGA, contains ABO blood group trisaccharide glycans with an ethylazido aglycone conjugated to groups of ten multi-barcoded M13 particles carrying dibenzocyclooctyne (DBCO) on p8 proteins. ELISA clonal binding assays to anti-A/B antibodies confirmed the functional performance of ABO-clones and aligned with next-generation sequencing (NGS) of the mixed clones. Multiple DNA-barcoded technical replicates in LiGA allow for quantification of reproducibility and robustness as determined by the Z'-score using NGS. We then tested ABO-LiGA for specific detection of IgG and IgM anti-A and anti-B IgG and IgM antibodies in human serum samples. Comparison of antibody binding responses in sera from 31 healthy donors to ABO-LiGA with an ABO-Luminex-based method revealed consistent responses to LiGA-ABO but also minor deficiencies of ABO-LiGA such as low robustness of the current assay format and a limited ability to detect low intensity antibody responses. Some results point to undesired interactions of serum antibodies with small-footprint glycans conjugated to phage via the bulky DBCO moiety. This report illuminates the path for future development of LiGA-based serological assays and suggests the need to develop alternative methods for conjugating glycans to phage to avoid liabilities of the hydrophobic DBCO moiety.
我们测试了多重液相聚糖阵列（LiGA）技术在血清学检测中的性能。具体而言，我们使用LiGA检测人血清中的ABO血型抗体。这种我们命名为ABO-LiGA的LiGA包含ABO血型三糖聚糖，其带有与十个多条形码M13颗粒基团共轭的乙基叠氮苷元，这些颗粒在p8蛋白上携带二苯并环辛炔（DBCO）。针对抗A/B抗体的ELISA克隆结合试验证实了ABO克隆的功能性能，并与混合克隆的下一代测序（NGS）结果一致。LiGA中多个DNA条形码技术重复样本可通过NGS使用Z'分数来量化可重复性和稳健性。然后，我们测试了ABO-LiGA对人血清样本中IgG和IgM抗A和抗B IgG及IgM抗体的特异性检测。将31名健康供体血清与ABO-LiGA的抗体结合反应与基于ABO-Luminex的方法进行比较，结果显示对LiGA-ABO有一致反应，但ABO-LiGA也存在一些小缺陷，如当前检测形式的稳健性较低以及检测低强度抗体反应的能力有限。一些结果表明血清抗体与通过庞大的DBCO部分与噬菌体共轭的小尺寸聚糖存在不良相互作用。本报告阐明了基于LiGA的血清学检测未来的发展方向，并表明需要开发将聚糖与噬菌体共轭的替代方法，以避免疏水性DBCO部分带来的问题。
