Measurement of human urokinase activity in plasma by using mono-specific antibody-conjugated paper disk.
Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4B. The purified enzyme was of a high molecular weight form (molecular weight of 53,000). This preparation was utilized as an antigen to immunize rabbits; the obtained antibody showed a high specificity against HUK. The antibody was conjugated to CNBr-activated paper disks. The antibody-conjugated paper disk and a fluorogenic peptide substrate, glutaryl-Gly-Arg-4-methyl-coumarine-7-amide, were used to measure urokinase (UK) activity in plasma. The calibration curve obtained by the proposed method passed through the origin and was linear in the range of 0-0.16 IU of HUK. The incubation of HUK with an excess amount of alpha 2-macroglobulin at 37 degrees C for 3 h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with alpha 1-antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37 degrees C decreased the activity as a function of time. However, when the antibody-conjugated paper disk was used for the immunoreaction to HUK in plasma at 4 degrees C, no decrease of UK activity was observed. The plasma decay curve of UK activity after a single intravenous (i.v.) injection of HUK into a rabbit (12,000 IU/kg) indicated bi-exponential kinetics by using this assay method. The rate constants of the alpha and beta phases were 0.120 +/- 0.020 and 0.021 +/- 0.002 min-1, respectively. These result suggest that the proposed method is useful for measuring UK activity in plasma of patients with intravascular coagulation after i.v. administration of UK.
D000818:Animals; D000911:Antibodies, Monoclonal; D000990:Antithrombin III / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010209:Paper; D011817:Rabbits; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000097:blood*; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; D000990:Antithrombin III; D014568:Urokinase-Type Plasminogen Activator