Development of an ELISA method for testing VWF ristocetin cofactor activity with improved sensitivity and reliability in the diagnosis of von Willebrand disease.

OBJECTIVES: Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand's disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time-consuming with suboptimal interassay and intra-assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis. METHODS: We have established a sensitive VWF:RCo-ELISA method using a monoclonal antibody, SZ-151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP-conjugated rabbit anti-human VWF IgG. We tested the VWF:RCo level by this VWF:RCo-ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody. RESULTS: The sensitivity of VWF:RCo-ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra-assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3-56.9), 4.8 (0.6-11.8), 8.6 (1.6-19.7), 3.9 (1.0-6.8), 1.0 (0.5-1.6), and 91.5 (47.3-169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo/VWF:Ag are 0.83 (0.70-1.16), 0.27 (0.08-0.58), 0.31 (0.15-0.40), 0.18 (0.14-0.21), 0.52 (0.13-1.19), and 0.92 (0.62-1.26). CONCLUSION: The VWF:RCo-ELISA using monoclonal anti-rfGPIbα antibody SZ-151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.