Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.
We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.
INSERM U 353, Hôpital Saint Louis, Paris, France.
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Morandi
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Edelman
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Legrand
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Legrand
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D015153:Blotting, Western; D004730:Endothelium, Vascular / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology*; D015539:Platelet Activation / Q000187:drug effects; D011994:Recombinant Proteins / Q000276:immunology; D013917:Thrombin / Q000494:pharmacology; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D000941:Antigens; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019699:Thrombospondins; D006493:Heparin; D013917:Thrombin