A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG

• Published in: Glycoconjugate journal Vol. 37; no. 6; pp. 691 - 702
• Main Authors: Rebello, Osmond D., Gardner, Richard A., Urbanowicz, Paulina A., Bolam, David N., Crouch, Lucy I., Falck, David, Spencer, Daniel I. R.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-12-2020; Springer Nature B.V
• Subjects: Antibodies; Biochemistry; Biomedical and Life Sciences; Chromatography; Dehydrogenases; Galactose; Glycoproteins; Glycosylation; Health care; Immunoglobulin G; Inflammatory bowel disease; Inflammatory diseases; Laboratories; Lectins; Life Sciences; Monosaccharides; Original; Original Article; Pathology; Glycomics; Plate assay; Glycosidase; Antibodies; Glycans; HPLC-FLD-MS; Sialylation; Galactosylation
• ISSN: 0282-0080; 1573-4986
• DOI: 10.1007/s10719-020-09953-9

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson’s r correlation coefficient of 0.8208 between the two methods.