Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.
Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.
Faculty of Biology, University of Konstanz, Germany.
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I
Siegle
R
R
Nüsing
R
R
Brugger
R
R
Sprenger
R
R
Zecher
V
V
Ullrich
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001011:Aorta / Q000201:enzymology / Q000648:ultrastructure; D015153:Blotting, Western; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D006801:Humans; D007163:Immunosorbent Techniques; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000032:analysis* / Q000276:immunology*; D008861:Microsomes / Q000201:enzymology; D012680:Sensitivity and Specificity; D013552:Swine; D014018:Tissue Distribution; D014471:Umbilical Veins / Q000201:enzymology
D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase