National Health Laboratory Services, Universitas Hospital, South Africa.
Department of Haematology and Cell Biology, University of the Free State, Bloemfontein 9301, South Africa.
Biomed Res Int. 2018 May 24;2018:6232091. doi: 10.1155/2018/6232091. eCollection 2018.
The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay.
We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay.
Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma.
Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations.
由于疾病的异质性，血管性血友病的诊断较为复杂。约 80%的血管性血友病患者被诊断为血管性血友病因子（VWF）定量缺陷，其中 50%是由于 VWF 清除率增加所致。这些患者对首选治疗药物去氨加压素（DDAVP）反应不佳，因为其疗效降低。VWF 前肽与成熟 VWF 抗原的比值用于诊断这些患者。商业 VWF 前肽检测试剂盒在发展中国家过于昂贵。在这项研究中，我们开发了一种具有成本效益的 ELISA 检测试剂盒。
我们首先在酵母上展示 VWF 前肽。通过噬菌体展示技术，针对展示的 VWF 前肽筛选抗体片段。这些抗体被用于开发一种具有成本效益的 VWF 前肽检测试剂盒，并与商业 VWF 前肽检测试剂盒进行比较。
其中两个抗体片段特异性结合 VWF 前肽，而不与用于表达前肽的酵母结合。这些纯化的抗体片段能够检测正常血浆中的 VWF 前肽。
与商业试剂盒相比，我们的检测试剂盒表现良好。它还显示出在特别是较低的血浆浓度下对 VWF 前肽具有更高的结合亲和力。
