Characterization of recombinant rat plasminogen activator inhibitor-1 and development of immunological tools for its quantitation.
Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Elevated plasma levels of PAI-1 have been associated with several important thrombotic diseases. A large number of studies have demonstrated that rats are suitable for in vivo investigations on thrombolysis and fibronolysis. In this study, we have expressed, in Escherichia coli, purified and characterized recombinant rat PAI-1 in comparison with human PAI-1. Subsequently this material was used to raise monoclonal antibodies using the hybridoma technology. Characterization of purified recombinant rat PAI-1 revealed that its functional and biochemical properties are similar to those of human PAI-1. Two fusions, with spleen cells from mice immunized with recombinant rat PAI-1, yielded 118 positive hybridomas. From these, 36 monoclonal antibodies were purified and evaluated for their applicability in the construction of sandwich-type ELISAs. Out of 860 combinations tested, 2 combinations were selected for the measurement of rat PAI-1 (antigen and activity) in biological samples (e.g., plasma, platelet lysates, cell-culture media, ...).
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Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Verheyen
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Knockaert
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Declerck
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PJ
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins