Validation of a von willebrand factor propeptide assay
Maleka, Rethabile Brigette
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Von Willebrand disease is the most common inherited bleeding disorder caused by a deficiency or defect in von Willebrand factor. Quantitative defects of von Willebrand factor include, type 1 von Willebrand disease (partial deficiency of von Willebrand factor) and type 3 von Willebrand disease (complete deficiency of von Willebrand factor). Type 2 von Willebrand disease includes all qualitative defects of von Willebrand factor. Type 1 von Willebrand disease is either due to decreased synthesis and secretion or increased clearance of von Willebrand factor from plasma. It is essential to diagnose individuals with an increased clearance rate of von Willebrand factor, as the treatment of these patients with 1-deamino-8-D-arginine vasopressin is not effective. The ratio between the von Willebrand factor propeptide and the von Willebrand factor antigen is used to identify conditions of reduced half-life, such as type 1 von Willebrand disease with increased clearance. Currently, there is only one commercial assay available to measure von Willebrand factor propeptide levels. This assay is not only too expensive to be used in developing countries but is also very time consuming. The von Willebrand factor propeptide protein assay is an expensive test as it uses monoclonal antibodies. Mammalian cells are commonly used for the expression of monoclonal antibodies. The production of monoclonal antibodies is expensive. With this research an effort was made to develop more cost-effective and more rapid assays to determine the von Willebrand factor propeptide levels in patient’s plasma. The aim of this study was to therefore validate a von Willebrand factor propeptide assay. First, two single chain variable fragments that bind to the von Willebrand factor propeptide were expressed by yeast. The von Willebrand factor propeptide protein was also expressed, as it is not commercially available. However, the expression of the propeptide and the two single chain variable fragments were not successful. The von Willebrand factor propeptide protein is firstly too large and it also consists of 2 homologous cysteine-rich D domains. A primary bottleneck in recombinant protein production is the presence of the disulfide bond structure. We then produced two polyclonal antibodies against a truncated form of the von Willebrand factor propeptide. The two polyclonal antibodies could however only detect the truncated von Willebrand factor propeptide, but not the von Willebrand factor propeptide in plasma. The reduced antigenicity of the truncation affected the epitope construction. We then developed a lateral flow assay using commercial antibodies to the von Willebrand factor propeptide. Lateral flow assays are low cost detection devices that are simple to use, rapid and portable. In the rapid von Willebrand factor propeptide lateral flow assay, a monoclonal and polyclonal clonal antibody was used. The polyclonal antibody did not bind specific to the von Willebrand factor propeptide as it can bind to both the full-length von Willebrand factor protein and to the von Willebrand factor propeptide. Polyclonal antibodies show higher cross reactivity. This assay could therefore not be validated, as it was not specific for the VWF propeptide. Lastly, a rapid enzyme-linked immunosorbent assay using the commercial antibody pair clone CLB-Pro 35 and CLB-Pro 14.3 was developed and validated. This rapid assay has equal sensitivity and precision as the commercial method and can be used to diagnose patients with increased von Willebrand factor clearance.