Format

Send to

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

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

Author information

1
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States.
2
Department of Biochemistry, Oxford University, Oxford, United Kingdom.
3
Thayer School of Engineering, Dartmouth College, Hanover, NH, United States.
4
Department of Neurology, Brigham and Women's Hospital, Boston, MA, United States.
5
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, United States.
6
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States. Electronic address: galter@partners.org.

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

PMID:
25523925
PMCID:
PMC5054724
DOI:
10.1016/j.jim.2014.12.004
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center