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A novel application of multi-wavelength TIRF spectroscopy for real time monitoring of antithrombin interactions with immobilized heparin
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
2006 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 21, no 10, 1973-80 p.Article in journal (Refereed) Published
Abstract [en]

Real time interactions of antithrombin (AT) with Corline Heparin Surfaces (CHS) with one and two layers of heparin conjugate have been examined using a multi-wavelength TIRF spectroscopy technique with continuous flow. Fluorescently labeled AT, adsorbed from citrated human blood plasma, showed significantly higher signals on CHS compared to the cationic surface used to attach the heparin conjugate. The AT binding to CHS was very stable, also after exposure to soluble heparin at a concentration of 1.5 IU/mL. Only a few percent of the bound AT were displaced from the surfaces by AT present in plasma after long-term exposure to plasma. In contrast, larger amounts of the freshly added AT had adsorbed to the surfaces, especially to the surface with two layers of heparin conjugate, indicating the presence of unsaturated AT binding sites. The amount of AT bound to the different surfaces was quantified after elution using an enzyme immunoassay (EIA). Characteristic emission spectra of proteins and fluorophores of labeled proteins, obtained at the surfaces after a long-term exposure to plasma, confirmed their presence at the surfaces. The multi-wavelength TIRF technique proved to be a useful tool when combined with other techniques to study the time course of interactions of fluorescently labeled proteins with biomaterials, even in a complex environment such as plasma.

Place, publisher, year, edition, pages
2006. Vol. 21, no 10, 1973-80 p.
Keyword [en]
Antithrombin, Blood compatibility, Heparinized surfaces, TIRF
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-124310DOI: 10.1016/j.bios.2005.10.001PubMedID: 16289605OAI: oai:DiVA.org:uu-124310DiVA: diva2:317325
Available from: 2010-05-04 Created: 2010-05-03 Last updated: 2017-12-12Bibliographically approved

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