The evaluation of factor VIII binding activity of von Willebrand factor by means of an ELISA method: significance and practical implications.
One of the functions of von Willebrand factor (vWF) is to serve as a carrier of clotting factor VIII (FVIII). Deficiency of this function results in the von Willebrand disease (vWD) variant type 2N, which resembles hemophilia A. We describe a new sandwich enzyme-linked immunosorbent assay (ELISA) to study the ability of vWF to bind exogenous recombinant FVIII (rFVIII), in which anti-vWF-coated plates are incubated with plasma vWF, followed by exogenous FVIII and a peroxidase-coupled anti-FVIII antibody. Dose-response curves obtained using normal plasma vWF and purified normal vWF revealed a hyperbolic relationship between the optical density and the vWF concentration. The assay allows the quantification of FVIII binding with values expressed in U/dL; 100 U/dL was the amount present in normal plasma. The sensitivity and specificity of the method are demonstrated by its ability to measure binding levels as low as 1 to 2 U/dL and the fact that no FVIII binding was observed using plasma known to contain less than 1 U/dL vWF. To verify the accuracy of the assay, three patients with type 2N vWD with characterized vWF gene mutations were studied using an existing chromogenic assay and our ELISA. A patient who was homozygous for the R53W mutation and had no FVIII binding capacity according to the chromogenic method showed undetectable FVIII binding by ELISA. The remaining two patients, one who was homozygous for the R91Q mutation and one with compound heterozygosity for the R91Q and R53W mutations, showed markedly decreased FVIII binding by the chromogenic method and yielded ELISA values ranging from 4 to 8 U/dL. Therefore, although the two methods produce qualitatively similar results, the ELISA method offers the advantage of allowing quantification of the FVIII binding function. FVIII binding was also analyzed in 20 patients with type 1 vWD; we found a decrease of FVIII binding that was proportionate to the decrease in vWF levels, showing a normal FVIII binding activity/vWF molecule ratio. We define the binding activity measured by this assay as vWF:FVIII binding activity and propose its use in the functional analysis of vWF.
Casonato
A
A
University of Padua Medical School, Institute of Medical Semeiotics, Italy.
Pontara
E
E
Zerbinati
P
P
Zucchetto
A
A
Girolami
A
A
D000328:Adult; D000368:Aged; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D014842:von Willebrand Diseases / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII