Immune Profiling and Biomarker Discovery Using a Multiplexed Antibody Fc Characterization Platform
Author(s)
Saha, Anushka
Advisor(s)
Sarkar, Aniruddh
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Abstract
Antibodies secreted by plasma cells play a significant role in disease outcomes and encode precise information due to the tunable structure of both the Fab and the crystallizable fragment (Fc) region. Several studies measure antigen-specific antibody titers to report changes within the Fab region, yet the Fc region, responsible for communicating with host immune cells to modulate function, is often unmeasured. This thesis characterizes the Fc-specific changes in serum antibodies to improve diagnostics and gain insight into immune pathways underlying disease outcomes. Post-translation, antibodies are modified with sugars, or N-glycans, which affect binding to downstream immune cells due to effects on the protein structure or charge, altering its function. Current gold standard methods to measure protein glycosylation are mass spectrometry with high-performance liquid chromatography (LC-MS) or capillary electrophoresis, which can be nearly impossible with small volumes of samples. MS requires isolation of high concentrations of target protein and an hour runtime for a single sample. This remains the bottleneck in glycoprofiling limited patient sample volumes from diseases which still lack clear diagnostics such as Schistosomiasis and Antibody-mediated rejection (ABMR). Our solution to probe antigen-specific glycosylation at a high throughput and low-cost is to couple antigens of interest to microspheres, apply patient serum, and use plant-based lectins which bind to N-linked glycans. My research uses beads coated with disease-specific antigens and unique Fc-binding probes including subtypes, Fcgamma receptors, and lectins RCAI and SNA to characterize antibody profiles of patient serum samples using flow cytometry; computational analysis methods are then applied to the data to identify key antibody features which classify disease from no disease.
In this thesis, I will address two aims:
1. Optimize a multiplexed binding assay to detect antibody glycosylation as an alternative to mass spectrometry using fluorescently labeled lectins.
2. Apply this method to measure antibody N-linked glycosylation and other tunable Fc properties to define robust biomarkers from clinical sample cohorts in Schistosomiasis and ABMR.
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Date
2023-05-02
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