Multiplexed Liquid Glycan Array (LiGA) for serological assays

• Author / Creator

  Revathi Reddy

• Human serum has a diverse pool of anti-glycan antibodies generated as an immune response to glycans presented by pathogens, tumor cells and vaccines. Low concentrations of antigens are often difficult to detect which renders anti-glycan antibodies as potential biomarkers for disease diagnosis and prognosis. Traditional approaches to epitope discovery do not account for an important event - post translational modification of proteins. There is an ever growing need to identify new structural classes of serum biomarkers that will serve as disease diagnostic and prognostic tools. Glycan arrays are an established high-throughput tool that allows characterization of the interactions between glycans and glycan binding receptors (proteins, antibodies). One of the drawbacks of conventional “solid” printed array is the necessity for specialized DNA-array reader instruments that are no longer available in most research and clinical laboratories. Most questions previously answered by DNA-array and reader technologies are now answered by deep-sequencing of DNA. Building on this change, we develop a multiplexed “liquid” glycan array, which encodes the same information as traditional solid glycan array but can be decoded and read by deep-sequencing technology. As a proof of principle, we created phage-glycan conjugates that target the well-recognized IgM blood group ABO antibodies that bind to A, B or H antigen displayed on the surface of erythrocytes. Panning of ABO-phage-glyco conjugates on ABO antibodies coated on 96 well plates and Illumina sequencing of the recovered phage DNA unveils the preferences of the antibody for specific glycan structure and its presentation (density) on phage. To complement the expensive deep sequencing, we also develop a qPCR assay to track glycan specific DNA barcodes. These experiments were further extrapolated to the more complex human serum for the detection of both naturally occurring IgG and IgM anti-glycan antibodies.

• Subjects / Keywords

  • Serological assays
  • Glycan Array
  • Multiplexing
  • Blood group glycans

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-x5j3-yr44

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.