Nori Porcine VWF ELISA Kit Summary

Alternative names for VWF: Von Willebrand factor

Alternative names for porcine: Pig, swine

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Von Willebrand factor (VWF) is a large multimeric blood glycoprotein crucial for blood clotting that promotes primary hemostasis, specifically, platelet adhesion. It is produced by endothelial cells and megakaryocytes to help platelets adhere to injured blood vessel walls. VWF is essential for primary hemostasis. It binds to collagen at vascular injury sites, recruits platelets, and carries coagulation factor VIII (FVIII), protecting it from premature degradation. VWF plays a major role in blood coagulation and is involved in the formation of blood vessels themselves. It forms large multimers that act as shear-sensitive “force sensors” in the bloodstream. Deficiency or defects in this protein cause von Willebrand disease (VWD), a common bleeding disorder. VWF is involved in many other diseases, including thrombotic thrombocytopenic purpura, Heyde’s syndrome, and possibly hemolytic–uremic syndrome.^[1] Increased plasma levels in many cardiovascular, neoplastic, metabolic (e.g. diabetes), and connective tissue diseases are presumed to arise from adverse changes to the endothelium, and may predict an increased risk of thrombosis.^[2] Platelet adhesion is mainly mediated via interactions with VWF, which acts as a bridge between the platelet surface receptor glycoprotein Ib (GpIb) and the exposed collagen after vascular injury. Genetic deficiencies of VWF or GpIb (Bernard–Soulier syndrome) result in bleeding disorders. Most cases of vWD are hereditary, but abnormalities of VWF may be acquired; aortic valve stenosis, for instance, has been linked to vWD type IIA, causing gastrointestinal bleeding – an association known as Heyde’s syndrome.^[3] High plasma VWF levels were found to be an independent predictor of major bleeding in anticoagulated atrial fibrillation patients.^[4] VWF is a marker of endothelial dysfunction, and is consistently elevated in atrial fibrillation, associated with adverse outcomes.^[5]

References

1. Sadler JE(1998). Annual Review of Biochemistry. 67 (1): 395–424.
2. Shahidi M (2017). Advances in Experimental Medicine and Biology. Vol. 906. pp. 285–306.
3. Vincentelli A, et al. (2003). The New England Journal of Medicine. 349 (4): 343–349.
4. Roldán V, et al. (2011) Journal of the American College of Cardiology. 57 (25): 2496–2504.
5. Khan AA, et al. (2020). Annals of Medicine. 52 (1–2): 1–11.