An IgG-Optimized Enzyme-Linked Lectin Assay (ELLA) for Quantitative Analysis of Immunoglobulin Glycosylation
Wiggins, C. D.; Lauffenburger, D. A.
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Antibody Fc glycosylation is modulated in a variety of disease and immune response contexts, altering downstream functional responses including antibody-dependent cellular cytotoxicity through modified immune cell Fc receptor binding. Accessible, high-throughput glycosylation assays such as enzyme-linked lectin assays (ELLAs) are essential to advance understanding of glycosylation regulation and function. However, current ELLA protocols lack standardization and optimization, and results are reported out in arbitrary absorbance units, limiting reproducibility and cross-study comparability. We developed an optimized multi-lectin parallel ELLA with three specific improvements: systematic optimization of incubation times and reagent concentrations; incorporation of Protein A for IgG specificity; and use of commercially available bovine fetuin B as a quantitative surrogate standard for cross-study reproducibility. Our panel of 8 lectins, SNA, RCA, LCA, PHA-E, PHA-L, MAL-I, WGA, and DSL, cover the major IgG glycoforms. We demonstrate that our ELLA panel can reveal biologically relevant cytokine-induced plasticity of IgG glycosylation profiles in immortalized B cells.
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