Measurement of Human Urokinase Activity in Plasma by Using Mono-Specific Antibody-Conjugated Paper Disk

抄録

Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4 B. 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 α₂-macroglobulin at 37°C for 3h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with α₁ antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37°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°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 α and β phases were 0.120±0.020 and 0.021±0.002min^-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.