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Prepublished online as a Blood First Edition Paper on March 27, 2003; DOI 10.1182/blood-2002-11-3497. This Article Full Text Full Text (PDF) All Versions of this Article: 2002-11-3497v1 102/2/718    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Watkins, N. A. Articles by Hillery, C. A. Search for Related Content PubMed PubMed Citation Articles by Watkins, N. A. Articles by Hillery, C. A. Related Collections Red Cells Cell Adhesion and Motility Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 July 2003, Vol. 102, No. 2, pp. 718-724 RED CELLS Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion Nicholas A. Watkins, Lily M. Du, J. Paul Scott, Willem H. Ouwehand, and Cheryl A. Hillery From the Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI; the Blood Research Institute, the Blood Center of Southeastern Wisconsin, Milwaukee, WI; and the Department of Haematology, University of Cambridge and National Blood Service, Cambridge, United Kingdom The enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: C. G. Jakobsen, N. Rasmussen, A.-V. Laenkholm, and H. J. Ditzel Phage Display Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78 Cancer Res., October 1, 2007; 67(19): 9507 - 9517. [Abstract] [Full Text] [PDF] K A Smith, P N Nelson, P Warren, S J Astley, P G Murray, and J Greenman Demystified...recombinant antibodies. J. Clin. Pathol., September 1, 2004; 57(9): 912 - 917. [Abstract] [Full Text] [PDF]

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