Production and characterization of monoclonal antibodies against conservedepitopes of P-selectin (CD62P)

P-selectin (CD62P) is an adhesion molecule expressed on the activated endot helium and activated platelets that is involved in the initial attachment o f leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodi es (mAbs) and P-selectin-deficient mice have shown that P-selectin is a pot ential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology . The aim of this study was to generate a novel panel of anti-P-selectin mA bs against the conserved epitopes present in several animal species. To pro duce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recogn ize human P-selectin were obtained. Individual mAbs that bound to human, ra t, mouse, rabbit and pig activated platelets were characterized by flow-cyt ometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.-KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12 ) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P -sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL6 0 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MBb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELIS A assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure s oluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are uniq ue since they recognize common epitopes conserved during mammalian evolutio n and they may be useful for studying P-selectin function in inflammatory m odels in various species.