A monoclonal antibody against human urokinase: characterization of the epitope and its localization in human kidney.
Monoclonal antibodies against human plasminogen activator urokinase have been produced. A G62 hybridoma-producing antibody (IgG) was purified on a DEAE-cellulose column, and it proved useful for the measurement, identification and purification of antigens that had approximate molecular weights of 55- and 33-Kdaltons. For immunochemical measurements and purification, a competitive enzyme-linked immunosorbent assay (ELISA) and affinity chromatography using antibody-immobilized Sepharose 4B were developed. The ELISA has sensitivity to 20 p mole antigen molecules. The binding capacity of the antigen on the affinity column was evaluated on SDS-polyacrylamide slab gels as well as by fibrin autography and ELISA. Results showed that there was quantitative purification with no loss of enzyme activity in the one-step procedure. Western blotting and affinity binding showed antigenic bands with apparent molecular weights of 55- and 33-Kdaltons. Because the 55-Kdalton form contains 33- and 22-Kdalton components connected by a disulfide bond, the epitope domain is present on the 33-Kdalton chain. Using this antibody, we examined human kidney sections by direct immunofluorescence to locate the antigen. It was found in epithelial cells convoluted segments, in glomerulus cells and in capillary endothelial cells, evidence that renal tubular cells synthesize the antigen which then is secreted in urine.
D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D013861:Thiocyanates; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D014568:Urokinase-Type Plasminogen Activator; D016650:Fluorescein-5-isothiocyanate