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      The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay

      research-article
      1 , 2 , , 2 , 3 , 3
      BioMed Research International
      Hindawi

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          Abstract

          Background

          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.

          Methods

          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.

          Results

          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.

          Conclusion

          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.

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          Most cited references21

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          Advancement and applications of peptide phage display technology in biomedical science

          Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.
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            Protein A chromatography for antibody purification.

            Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.
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              Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris

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                Author and article information

                Contributors
                Journal
                Biomed Res Int
                Biomed Res Int
                BMRI
                BioMed Research International
                Hindawi
                2314-6133
                2314-6141
                2018
                24 May 2018
                : 2018
                : 6232091
                Affiliations
                1National Health Laboratory Services, Universitas Hospital, South Africa
                2Department of Haematology and Cell Biology, University of the Free State, Bloemfontein 9301, South Africa
                3Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein 9301, South Africa
                Author notes

                Academic Editor: Wolfgang Miesbach

                Author information
                http://orcid.org/0000-0002-5202-7702
                Article
                10.1155/2018/6232091
                5994315
                29992156
                503657fd-33a4-47e0-bc47-771394a135d2
                Copyright © 2018 S. M. Meiring et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 24 October 2017
                : 12 March 2018
                : 25 March 2018
                Funding
                Funded by: Technology Innovation Agency of South Africa
                Categories
                Research Article

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