PB0181 - A new enzyme-linked immunosorbent assay to quantify glycosylated fibrinogen in plasma

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Nicoline Daugaard, MSc

MSc, Ph.d.-student
Unit for Thrombosis Research, Department of Clinical Biochemistry, University Hospital of Southern Denmark, Esbjerg, Denmark
Esbjerg, Syddanmark, Denmark

Co-Author(s)

AB

Anna-Marie Bloch Münster

Associate Professor, PhD
Unit for Thrombosis Research, Department of Clinical Biochemistry, University Hospital of Southern Denmark, Esbjerg, Denmark
Esbjerg, Syddanmark, Denmark
EB

Else-Marie Bladbjerg

Professor, MSc, PhD
Unit for Thrombosis Research, Department of Clinical Biochemistry, University Hospital of Southern Denmark, Esbjerg
Esbjerg, Syddanmark, Denmark
Moniek de Maat

Professor, MSc, PhD
Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
AL

Anette Larsen

Laboratory technician
Unit for Thrombosis Research, Department of Clinical Biochemistry, University Hospital of Southern Denmark
Esbjerg, Syddanmark, Denmark
YP

Yaseelan Palarasah

Associate professor, MSc, PhD
Unit for Thrombosis Research, Department of Clinical Biochemistry, University Hospital of Southern Denmark
Esbjerg, Syddanmark, Denmark

Background: Stroke is a major cause of death globally, and diabetes patients are at higher risk of developing a stroke than non-diabetes individuals. The reason for this is unclear, but plasma fibrinogen seems to be involved due to its central role in clot formation and inflammation. Fibrinogen will be more glycosylated in the presence of high blood glucose levels, which are often present in diabetes patients. Glycosylated fibrinogen modulates clot structure, making it more resistant to lysis, but only little is known about the effects of glycosylation and the risk of stroke.

Aims: To develop an enzyme linked immunosorbent assay (ELISA) for quantification of glycosylated (sialylated) fibrinogen in human plasma.

Methods: In brief, a microtiter plate is coated with a polyclonal rabbit anti-human fibrinogen antibody (Dako, Glostrup, Denmark) and the plate is incubated with citrate plasma. Sialic acids on fibrinogen are detected by biotinylated sambucus nigra lectin ((SNL), Vector Laboratories, Newark, USA) and quantified using horseradish peroxidase (HRP)-conjugated streptavidin and 1-StepTM 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) substrate solution (Thermo Fisher Scientific, Rockford, USA). The plate is read at 405 nm using a microplate reader and the color intensity is proportional to the amount of sialic acids on fibrinogen. The assay is calibrated against a citrate plasma pool from healthy individuals, and the level of sialic acids is expressed relative to the fibrinogen concentration. Next, the method will be validated and applied on 153 samples from diabetes patients with stroke, 153 diabetes patients without stroke, and 120 healthy controls.

Results: Results will be presented at the ISTH congress.

Conclusion(s): Informed consent is obtained from the patients and the study is approved by the Danish Research Ethics Committee. This work was supported by a grant from Steno Diabetes Center Odense, funded by the Novo Nordisk Foundation.