Journal of Immunological Methods 2015-02-01

A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis.

Alison E Mahan, Jacquelynne Tedesco, Kendall Dionne, Kavitha Baruah, Hao D Cheng, Philip L De Jager, Dan H Barouch, Todd Suscovich, Margaret Ackerman, Max Crispin, Galit Alter

Index: J. Immunol. Methods 417 , 34-44, (2015)

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Abstract

The N-glycan of the IgG constant region (Fc) plays a central role in tuning and directing multiple antibody functions in vivo, including antibody-dependent cellular cytotoxicity, complement deposition, and the regulation of inflammation, among others. However, traditional methods of N-glycan analysis, including HPLC and mass spectrometry, are technically challenging and ill suited to handle the large numbers of low concentration samples analyzed in clinical or animal studies of the N-glycosylation of polyclonal IgG. Here we describe a capillary electrophoresis-based technique to analyze plasma-derived polyclonal IgG-glycosylation quickly and accurately in a cost-effective, sensitive manner that is well suited for high-throughput analyses. Additionally, because a significant fraction of polyclonal IgG is glycosylated on both Fc and Fab domains, we developed an approach to separate and analyze domain-specific glycosylation in polyclonal human, rhesus and mouse IgGs. Overall, this protocol allows for the rapid, accurate, and sensitive analysis of Fc-specific IgG glycosylation, which is critical for population-level studies of how antibody glycosylation may vary in response to vaccination or infection, and across disease states ranging from autoimmunity to cancer in both clinical and animal studies. Copyright © 2014 Elsevier B.V. All rights reserved.


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