A Reliable and Reproducible ELISA Method to Measure Ristocetin Cofactor Activity of von Willebrand Factor

Abstract

SummaryThe ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIbα-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory antiGPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand’s disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter-and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 µl).

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

“…VWF antigen (VWF:Ag) levels in the plasma samples obtained from the in vitro LVAD systems, were quantified by ELISA as described. 30,31 Briefly, a 96-well microtiter plate was coated with 4.1 μg/ml of polyclonal anti-human VWF antibodies (Dako), blocked with a 3% milk powder solution and a 1/80 dilution of human or bovine plasma was added and the samples were further diluted by one half in PBS containing 0.3% milk. Next, bound VWF was detected using polyclonal anti-human VWF antibodies labeled with horseradish peroxidase (Dako).…”

“…VWF collagen binding activity (VWF:CB) was determined using ELISA as described. 32,33 Briefly, 25 μg/ml of human collagen type III (Sigma-Aldrich) for binding of human VWF or 25 μg/ml of human collagen type I (Sigma-Aldrich) for binding of bovine VWF was coated onto a 96-well microtiter plate and the plate was blocked with a 3% milk powder solution. Next, a 1/80 dilution of plasma was added and the samples were further diluted by half in PBS containing 0.3% milk.…”

Obstacles to Early Diagnosis and Treatment of Inherited von Willebrand Disease: Current Perspectives

“…24 However, traditional VWF:RCo assays using normal fresh or formalin-fixed platelets have low inter-laboratories reproducibility, 50 high coefficient of variation and poor sensitivity for very low VWF values. The optimal ristocetin concentration may be another limitation of the test, 51 as well as also the erroneous low levels in presence with p. P1467S and p.D1472H VWF polymorphisms. 52 The search for assays with less variability and increased sensitivity than VWF:RCo has prompted the development of new tests.…”