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    Validation of an automated ultraperformance liquid chromatography IgG N-glycan analytical method applicable to classical galactosaemia


    Colhoun, Hugh Owen and Treacy, Eileen P and MacMahon, Marguerite and Rudd, Pauline M. and Fitzgibbon, Maria and O'Flaherty, Roisin and Stepien, Karolina M (2018) Validation of an automated ultraperformance liquid chromatography IgG N-glycan analytical method applicable to classical galactosaemia. Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 55 (5). pp. 593-603. ISSN 1758-1001

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    Abstract

    Background: Classical galactosaemia (OMIM #230400) is a rare disorder of carbohydrate metabolism caused by deficiency of the galactose-1-phosphate uridyltransferase enzyme. The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems, remains poorly understood. Current clinical methods of biochemical monitoring lack precision and individualization with an identified need for improved biomarkers for this condition. Methods: We report the development and detailed validation of an automated ultraperformance liquid chromatography N-glycan analytical method of high peak resolution applied to galactose incorporation into human serum IgG. Samples are prepared on 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labelling and post-labelling clean-up with solid-phase extraction. Results: This method is shown to be accurate and precise with repeatability (cumulative coefficients of variation) of 2.0 and 8.5%, respectively, for G0/G1 and G0/G2 ratios. Both serum and processed N-glycan samples were found to be stable at room temperature and in freeze–thaw experiments. Conclusions: This high-throughput method of IgG galactose incorporation is robust, affordable and simple. This method is validated with the potential to apply as a biomarker for treatment outcomes for galactosaemia.

    Item Type: Article
    Keywords: Galactosaemia, method development, N-glycan glycoproteins, biomarkers, IgG
    Academic Unit: Faculty of Science and Engineering > Chemistry
    Item ID: 15037
    Identification Number: https://doi.org/10.1177/0004563218762957
    Depositing User: Roisin O'Flaherty
    Date Deposited: 16 Nov 2021 17:03
    Journal or Publication Title: Annals of Clinical Biochemistry: International Journal of Laboratory Medicine
    Publisher: SAGE Publications
    Refereed: Yes
    URI:

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