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Measurement of Neutral and Sialylated IgG n-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis

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Clinical Applications of Capillary Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1972))

Abstract

Modulations in immunoglobulin G (IgG) n-glycosylation have been observed in many human diseases including chronic inflammatory diseases such as rheumatoid arthritis and also cancer. In this chapter, we describe how to determine hypogalactosylation for clinical samples, namely the sample preparation of IgG n-glycans at Asn-297 as well as the measurement of neutral and sialylated n-glycans by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF).

This semiautomated protocol describes the isolation polyclonal antibodies from serum, the separation of IgG-Fc glycopeptides from IgG antigen-binding fragment by pepsin digestion. Afterward, enzymatically released IgG-Fc n-glycans are cleaned up using a polyaromatic adsorbent resin followed by carbon purification. Sialic acids are then derivatized prior to glycan labeling. As a result, the agalactosylated n-glycan A2 does not co-migrate with sialylated n-glycans, which refines the measurement of hypogalactosylation by CE-LIF.

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Acknowledgements

Authors acknowledge Peggy Thiele for her technical assistance.

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Authors and Affiliations

  1. Charité—Universitätsmedizin Berlin, Campus Virchow Klinikum, Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Berlin, Germany

    Christian Schwedler & Véronique Blanchard

Authors
  1. Christian Schwedler
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  2. Véronique Blanchard
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Correspondence to Véronique Blanchard .

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Editors and Affiliations

  1. Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA

    Terry M. Phillips

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Schwedler, C., Blanchard, V. (2019). Measurement of Neutral and Sialylated IgG n-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis. In: Phillips, T.M. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology, vol 1972. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9213-3_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9213-3_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9212-6

  • Online ISBN: 978-1-4939-9213-3

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