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

Abstract

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.

Total serum N-glycans mark visceral leishmaniasis in human infections with Leishmania infantum

“…In addition, for clinical implementation, blood glycome analysis will have to be transferred from high-end mass spectrometry to e.g., a microtiter plate assay. 31 , 41 , 45 …”

“…Here, we analyzed total serum N -glycosylation in Visceral leishmaniasis using a high-throughput technique. 39 , 40 , 41 We explored associations of total serum N -glycosylation and VL with regards to presence or absence of symptoms, severity of disease and post-treatment recovery to obtain insights into potential new markers for diagnosis and therapy monitoring.…”

Equal mixing time enables scale‐down and optimization of a CHO cell culture process using a shaken microbioreactor system

“…The N‐glycans from the IgG4 samples were released using PNGaseF, procainamide labelled and analyzed on a HILIC‐FLD‐MS platform as previously described. [ 20,21 ] Reagents used for N‐glycan release were obtained from the LudgerZyme PNGase F Release Kit (LZ‐rPNGaseF‐kit, Ludger). Briefly, cell‐ expressed IgG4 glycoproteins obtained using the different culture methods were dried down.…”

Detection of Sialic Acid and Imaging of Cell-Surface Glycan Using a Fluorescence–SERS Dual Probe

“…Chromatographic techniques, including gas chromatography (GC) and high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS), are employed commonly to distinguish among the derivatives of SA and other sugars . This differentiation can also be achieved by enzymatic assays. − However, they require expensive proteins and sophisticated biological tools. On the other hand, colorimetric and fluorometric assay mainly utilizes boronic acid derivatives, which are, of course, the most reliable and excellent candidates to bind with various sugars (Figure ).…”