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Shedding light on glycosylation: Analysis of complex carbohydrates using an affinity biosensor
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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).

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 49 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 675
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25678Local ID: 10054ISBN: 91-7219-970-9 (print)OAI: oai:DiVA.org:liu-25678DiVA: diva2:246226
Public defence
2001-05-18, Administrationsbyggnadens aula, Universitetssjukhuset, Linköping, 13:30 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2015-09-18Bibliographically approved
List of papers
1. Detection of low-molecular-weight heparin oligosaccharides (Fragmin™) using surface plasmon resonance
Open this publication in new window or tab >>Detection of low-molecular-weight heparin oligosaccharides (Fragmin™) using surface plasmon resonance
1998 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 11, no 1-6, 191-193 p.Article in journal (Refereed) Published
Abstract [en]

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide–protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin™) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore™), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.

Place, publisher, year, edition, pages
John Wiley and Sons, 1998
Keyword
surface plasmon resonance, low-molecular-weight heparin, oligosaccharides
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-75800 (URN)10.1002/(SICI)1099-1352(199812)11:1/6<191::AID-JMR421>3.0.CO;2-4 (DOI)000078789000043 ()
Available from: 2012-03-12 Created: 2012-03-12 Last updated: 2017-12-07Bibliographically approved
2. Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance
Open this publication in new window or tab >>Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance
2000 (English)In: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 17, no 5, 323-329 p.Article in journal (Refereed) Published
Abstract [en]

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25225 (URN)10.1023/A:1007169621518 (DOI)9664 (Local ID)9664 (Archive number)9664 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. A Lectin Immunosensor Technique for Determination of α1-Acid Glycoprotein Fucosylation
Open this publication in new window or tab >>A Lectin Immunosensor Technique for Determination of α1-Acid Glycoprotein Fucosylation
Show others...
2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 288, no 2, 216-224 p.Article in journal (Refereed) Published
Abstract [en]

The fucosylation of α1-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.

Keyword
a1-acid glycoprotein, Aleuria aurentia lectin, Fucose, Glycosylation, Surface plasmon resonance
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25224 (URN)10.1006/abio.2000.4905 (DOI)9663 (Local ID)9663 (Archive number)9663 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique
Open this publication in new window or tab >>Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique
2002 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 17, no 10, 883-891 p.Article in journal (Refereed) Published
Abstract [en]

A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze α1-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor β-1 (TGFβ1). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGFβ1 AGP fucosylation increased whereas AGP concentration decreased.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25226 (URN)10.1016/S0956-5663(02)00111-2 (DOI)9665 (Local ID)9665 (Archive number)9665 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved

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