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Vanderschaeghe D.,Unit for Molecular Glycobiology | Guttman A.,Debrecen University | Callewaert N.,Ghent University
Methods in Molecular Biology | Year: 2013

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward. © 2013 Springer Science+Business Media, LLC. Source


Vanderschaeghe D.,Unit for Molecular Glycobiology
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the γ-globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein L-agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liver fibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the γ-globulins (r = 0.2-0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test. Source


Vanderschaeghe D.,Unit for Molecular Glycobiology
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward. Source


Debruyne E.N.,Ghent University | Vanderschaeghe D.,Ghent University | Van Vlierberghe H.,Ghent University | Vanhecke A.,Unit for Molecular Glycobiology | And 2 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: Hepatocellular carcinoma (HCC) is a common and rapidly fatal cancer. Current diagnostic methods for HCC have poor sensitivity and specificity, are invasive, and carry risk for complications. Newer markers are needed to overcome these problems and allow diagnosis of HCC at an earlier stage. In view of known associations between glycosylation changes and liver disease, we focused on the serum glycoprotein hemopexin and the specific characteristics of this liver-synthesized glycoprotein. METHODS: We studied 49 healthy volunteers and 81 patients divided into the categories of fibrosis, cirrhosis, and HCC with cirrhosis. Hemopexin was purified from study participants' serum by use of heme agarose beads. The hemopexin N-glycan profile was determined by use of the DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis technique. RESULTS: We found that branching α-1,3-fucosylated multiantennary glycans on hemopexin were increased in theHCCgroup compared with the cirrhosis without HCC, fibrosis, and healthy volunteer groups, whereas nonmodified biantennary glycans decreased progressively across groups from fibrosis to the cirrhosis and HCC groups. Summarization of this information in a new marker, called the hemopexin glycan marker, enabled distinction of patients with HCC and cirrhosis from healthy volunteers and patients with fibrosis or cirrhosis with a sensitivity and specificity of 79% and 93%, respectively. CONCLUSIONS: This study demonstrated hemopexin to be a model protein for studying liver-specific N-glycosylation. The hemopexin glycan marker could be a valuable complementary test to α-fetoprotein measurements for detection of HCC in patients with cirrhosis. Additional study of its utility for diagnosis and follow-up is recommended. © 2010 American Association for Clinical Chemistry. Source


Vanderschaeghe D.,Unit for Molecular Glycobiology | Debruyne E.,Ghent University | Van Vlierberghe H.,Ghent University | Callewaert N.,Ghent University | Delanghe J.,Ghent University
Methods in Molecular Biology | Year: 2013

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the γ-globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein l -agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liverfibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the γ-globulins (r = 0.2- 0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test. © 2013 Springer Science+Business Media, LLC. Source

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