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

J Immunol Methods. 2015 Feb;417:34-44. doi: 10.1016/j.jim.2014.12.004. Epub 2014 Dec 15.

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.

Keywords: Capillary electrophoresis; Fc separation; Glycan analysis; IgG N-glycosylation.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Electrophoresis, Capillary / methods*
  • Glycosylation
  • High-Throughput Screening Assays*
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / metabolism*
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / metabolism*
  • Immunoglobulin G / analysis*
  • Immunoglobulin G / immunology
  • Macaca mulatta
  • Mice
  • Polysaccharides / analysis

Substances

  • Immunoglobulin Fab Fragments
  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Polysaccharides
  • glycosylated IgG