Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity.
Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.
Fischer
B E
BE
Byk Gulden, Dept. FMF, Konstanz, Germany.
Thomas
K B
KB
Dorner
F
F
D001681:Biological Assay / Q000379:methods*; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D014841:von Willebrand Factor / Q000032:analysis* / Q000378:metabolism*
D014841:von Willebrand Factor; D003094:Collagen