Shedding light on glycosylation : Analysis of complex carbohydrates using an affinity biosensor
Abstract: The main objective during the work described in this thesis has been to develop assays for stodies of complex carbohydrates in a clinical context. Complex carbohydrates, such as free or protein linked oligosaccharides, have been shown to have important biological functions. Furthermore, certain diseases have been associated with changes in oligosaccharide composition. We wanted to explore the potential of affinity biosensors to analyze such changes, since disease-specific carbohydrate stroctores might be useful in the diagnosis and prognosis of diseases.Throughout this work a surface plasmon based affinity biosensor instrument has been used. The instrmnent detects changes in refractive index close to a sensor surface. By immobilization of, e.g., an antibody at the sensor surface, the change in refractive index caused by antigen binding can be followed over time. The change in refractive index is proportional to the mass of bound antigen. This detection technique was used in the present work to quantitate free oligosaccharides as well as the presence of certain carbohydrate stroctores on glycoproteins.Two different types of assays for analysis of complex carbohydrates were developed. The first type of assay was a single step assay, where a carbohydrate-binding protein was immobilized at the sensor surface. Changes in response due to binding of free oligosaccharides or glycoproteins in samples were then used to quantitate the content of specific carbohydrate structores. The second type of assay was a sandwich assay, where antibodies directed to the glycoprotein to be stodied were immobilized at the sensor surface. This allowed the glycoprotein to be captored at the sensor surface from crude samples such as diluted blood plasma. The glycosylation of the captored glycoprotein was then analyzed by injection of a lectin over the sensor surface. The relative amount of the carbohydrate stroctore recognized by the lectin was determined by calculating a ratio between sensor responses for bound lectin and captored glycoprotein.The single step assay was used to quantitate Fragmin ® concentration, a low molecular weight heparin used as an anticoagulant. The single step assay was further used to analyze immunoglobulin G from rheumatoid arthritis patients and control individuals for oligosaccharide chains deficient in galactose, a carbohydrate structure associated with this disease. The sandwich type assay was used to follow changes in fucosylation of an acute-phase protein, α1-acid glycoprotein, in plasma from patients with severe burns. The same assay was also used to stndy the effect of cytokines on secretion and glycosylation of α1-acid glycoprotein from a human hepatoma cell line (HepG2).
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