Preparation And Application Of A Monoclonal Antibody Against Human Von Willebrand Factor

Von Willebrand Factor(VWF) is a multimer produced by endothelial cells and megakarocytes, being stored in the Weibel-Palade bodies of the endothelial cells and in the cc-granules of platelets. In plasma, vWF performs 2 main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries and protects coagulation factor VIII. One of the most common bleeding disorders, von Willebrand disease(vWD), is due to a quantitative or qualitative defect of vWF.Boneu et al were the first to propose the measurement of plasma vWF as an index of endothelial damage in vascular disease. The preparation of Monoclonal antibody against vWF will benefit the diagnosis and analysis of the vWF-related diseases. Therefore, a novel McAb against vWF was produced in our laboratory. vWF was purified from Factor VIII concentrates. Balb/c mice were immunized by intraperitoneal and subcutaneous injection of vWF. After fusing and screening, A murine McAb4E10 was produced, which raised aganist von Willebrand factor, belonged to IgG, subclass and had no reaction with fibronectin and fibrinogen. It was confirmed by immunofluorence method that McAb4E10 stained fixed cultured Human Umbilical Vascular Endothelial Cells (HUVECS) with a "dotted" staining pattern. McAb4E10 recognized all small and large blood vessels wall cells in all tissue sections detected by immunohistochemical staining, showing that antigen was expressed in endothelial cells lining both arteries and veins. And McAb4E10 also reacted selectively with HUVECS, while human leucocytes, red blood cells and inactive platelets remained negative. Western blotting demonstrated that McAb4E10 reacted with two antigen-bands whose molecular weight were 225kD and 180kD under the reducing condition, as did McAbSZ-29. And radioimmunoassy confirmed that McAb4E10, SZ-29 and SZ-34 recognized different domain of vWFprotein. McAb4E10 could provide us an effective and reliable analytic method in vWF-related diseases during several pathologic conditions.The expression of P-selectin and the secrection of vWF were increased by stimulating HUVEC within few minutes. So cellular inflammatory model was initially created. Binding sites for several physiologically important ligands have been localized in the vWF subunit sequence. The site of interaction with platelet GpIb has been localized to the A1-repeat. TwovWF domains, A1 and A3, have been shown to interact with collagen in a variety of different experimental models. The FVIII binding domain has been localized to repeats D prime and part of D3. The tetrapeptide sequence Arg-Gly-Asp-Ser (RGBS), which mediates the binding of VWF to the platelet glycoprotein IIb-IIIa complex of activated platelets, occurs near the C-terminus of the Cl-repeat. However we found McAb4E10 had no effect on the platelets aggregation vWF-collagen binding, platelets-vWF adhesion by platelet aggregation test, collagen binding assay and the method of staining the platelet by Rose Bengal.Several other compound have been shown to bind vWF, although the physiological significance of these additional interactions is not known. So the production of novel monoclonal antibody is an interesting approach for the identification of new binding sites of this molecule and may be a useful tool for diagnostic analysis.