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A NEW SIMPLE AND RELIABLE METHOD FOR INVESTIGATING THE AFFINITY BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR IN PLASMA SAMPLES
Abstract number: P-T-211
Karlman1 M., Karlsson1 A., Wiman1 B.
1Department of Clinical Chemistry, Karolinska University Hospital, Institution of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
How-to-cite Karlman M, Karlsson A, Wiman B. A NEW SIMPLE AND RELIABLE METHOD FOR INVESTIGATING THE AFFINITY BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR IN PLASMA SAMPLES. J Thromb Haemost 2007; 5 Supplement 2: P-T-211
Abstract
Introduction: There is a need for more reliable methods measuring the interaction between coagulation factor VIII (fVIII) and von Willebrand factor (vWf) in plasma samples, for use in the clinical routine. We have developed a method measuring the affinity between fVIII and vWf in plasma samples, utilizing an ELISA system.
Methods: For this purpose microtiter plates have been coated with a monoclonal antibody (ESH-8, reacting with the C2 domain, American Diagnostica) against factor VIII. Thereafter the wells were treated with rfVIII (Kogenate Bayer®, kindly supplied by Bayer Healthcare Nordic, courtesy of Christer Dybeck). After washing, diluted plasma samples were added and incubated for 1 h. Then HRP-conjugated antibodies against vWf were added and used for quantification of bound vWf.
Results: If the samples were diluted to give similar final concentrations of vWf, the bound vWf was proportional to the affinity between fVIII and vWf. The interaction was clearly decreased if the Ca2+ concentration in the mixture was higher than 0.1 mmol/L. The binding of vWf from different individuals with normal vWf concentrations and fVIII-binding (n = 15) was determined as 100% 7.4% (SD). Three patients with known von Willebrand disease, type 2N, all had severely diminished binding. The high reproducibility and precision of the method will allow us to identify individuals with only slightly reduced bindings. Kinetic analysis using plasmon surface resonance has been started.
Conclusions: A new simple and very reproducible method for analyzing the interaction between rfVIII and vWf in plasma samples has been developed. The interaction is very dependent on the Ca2+ concentration.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
Session Details
| Date: |
01/08/2007
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| Time: |
00:00-00:00
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| Session name: |
XXIst ISTH Congress |
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| Location: |
Oxford, UK |
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