A Ristocetin-Independent ELISA for the Measurement of VWF Activity.

Von Willebrand Disease (VWD) is a relatively common bleeding disorder characterized by both quantitative and qualitative deficiencies in von Willebrand Factor. The correct diagnosis and classification of VWD is made on the basis of a personal and family bleeding history and the results of a panel of laboratory tests. Evaluations of both VWF antigen level and function are required. Most commonly VWF function is evaluated based on VWF binding to platelets through the platelet surface glycoprotein GPIba in a platelet aggregation assay (VWF:RCo). The VWF:RCo assay requires the use of the antibiotic ristocetin to induce a conformational change in VWF which allows VWF to bind to GPIba on the surface of fresh or fixed platelets in the absence of VWF denaturation. When evaluating 172 normal controls, Flood et. al. (Blood, 2010) elegantly demonstrated that 63% of African-American controls and 17% of Caucasian controls had a single nucleotide polymorphism, D1472H, which altered the ability of these individuals to bind ristocetin and resulted in abnormal VWF:RCo results in the absence of any bleeding abnormalities.

GTI Diagnostics, Inc. (Waukesha, Wisconsin) has developed a ristocetin-independent fluorescent ELISA for the quantitative measurement of VWF activity in plasma, specifically VWF binding to immobilized GPIba. This VWF activity ELISA, referred to as the GTI IbCo Assay, uses microwells coated with recombinant GPIba containing two “gain-of-function” mutations. The mutant GPIba is capable of spontaneous binding to VWF in the absence of ristocetin or VWF denaturation (Flood et. al, Blood, 2010). A brief description of the assay follows. In the absence of ristocetin, calibrators, controls, and samples are added to the microwells and VWF binding to GPIba is allowed to take place. Unbound material is washed from the wells and a biotinylated anti-VWF detection antibody is added to the wells. The microwells are washed to remove any unbound detection antibody. A streptavidin-labeled horseradish peroxidase conjugate is added to the microwells and incubated. After a final wash step, a fluorescent peroxidase substrate is added to the wells. The reaction is quenched after 30 minutes and the fluorescence is read using an excitation wavelength between 315 and 340 nm and an emission wavelength between 370 and 470 nm. The reportable result (U/dL) of VWF binding to GPIba is determined from the standard curve. All assay incubation steps are 30 minutes and conducted at room temperature, therefore the assay can be completed in < 3 hours.

A small scale method comparison study was conducted on 11 plasma samples purchased from George King Bio-Medical, Inc. The plasma samples are Factor Assay ConTrol (FACT) samples and extensively characterized by George King Bio-Medical, Inc for 25 different coagulation parameters including VWF:RCo using a Chrono-Log platelet aggregometer and Helena Laboratories lyophilized platelets. The 11 samples were tested in the GTI IbCo Assay and the results obtained were compared to those obtained with the comparative method, in this case the VWF:RCo values provided by the supplier. For standardization of the GTI IbCo Assay, a IbCo value of 100 U/dL was assigned to the ISTH Secondary Coagulation Standard Lot #3. As illustrated in the table below, the samples tested included samples with varying levels VWF:RCo activity. When the results were compared, there was good correlation and agreement between the two methods. Larger method comparison studies will be conducted.

Based on this limited data set the GTI IbCo Assay demonstrated good correlation and agreement with the “gold standard” Ristocetin Cofactor Assay by platelet aggregation. The GTI IbCo Assay is able to measure the functional activity of VWF, specifically in regards to VWF binding to GPIba in the absence of ristocetin in a simple ELISA format. The GTI IbCo Assay removes the need for fresh or lyophilized platelets from the traditional assay and also eliminates the ristocetin dependence from the VWF activity measurement. By removing any dependence on ristocetin to induce the VWF to GPIba interaction we have also removed any possible assay discrepancies based on a patient's inability to interact with ristocetin.

Stapleton:GTI Diagnostics: Employment. Bub:GTI Diagnostics: Employment. Chance:GTI Diagnostics, Inc: Employment.