Technozym® uPA Combi Actibind® ELISA is an assay kit for the combined quantitative determination of concentration and activity of u-PA in human plasma. u-PA Actibind® assay is based on a catching antibody which does not interfere with the functional activity of u-PA. Following the binding of u-PA in the sample by the antibody, functional activity of bound u-PA is determined using Glu-Plasminogen and a low molecular weight plasmin substrate. After measuring the functional activity, this determination system is washed away and bound u-PA antigen is detected using a peroxidase-labeled monoclonal anti u-PA antibody which recognizes both free u-PA and u-PA inhibitor complexes.
The Technozym® Actibind® u-PA test measures u-PA functional activity, noncomplexed u-PA antigen and u-PA-inhibitor complexes. It is not affected by the presence of other plasminogen activators. The test system measures u-PA antigen in a range from 0-25 ng/ml and uPA activity from 0-2.5 IU/ml. The inter- and intra-assay variations are less than 10 % and 5 %, respectively.
- Plate + Plate Cover
12 x 8 well plastic microtitre strips precoated with a monoclonal anti-u-PA coating antibody in bicarbonate buffer,1% bovine serum albumin (BSA), (TC-Code GU).
1x lyophilized urokinase, calibrated against NIBSC 87/594 (TC-Code AQ).
1x conjugated polyclonal anti u-PA antibody (concentrated). (TC-Code AN).
- Dilution Buffer – (white cap)
1x 20 ml 2.5x concentrated (PBS, 1 % BSA, 20 mM EDTA, 10KIU/ml aprotinin, 20 mM Benzamidine) (TC-Code DC).
- POX Dilution Buffer – (white cap)
1x 12 ml PBS, 1 % BSA Ready to use (TC-Code DD).
- Substrate – (green cap)
1x 12 ml TMB (Tetramethylbenzidine) in substrate buffer containing H2O2. Ready to use (TC-Code KN).
- Stop Solution – (red cap)
1x 15 ml 0.5 M Sulphuric Acid (TC-Code KK). Ready to use.
- Wash Buffer – (blue cap)
1x 20 ml 12.5x concentrated (PBS, 0.5 %, Tween 20) (TC-Code BE).
- Plasminogen Activator Detection Mixture
1x lyophilized H-D-norleucyl-hexahydrotyrosyl lysine-p-nitroanilide diacetate salt and Gluplasminogen (TC Code BA).
- Detection Mixture Dilution Buffer (white cap)
1x 20 ml 50mM Tris, 12mM NaCl. Ready to use (TC-Code DA).
In the Actibind® u-PA test a monoclonal antibody which does not block u-PA functional activity is coated onto a microtitre plate and used to bind u-PA contained in plasma to the plate surface. Following an incubation period, non-bound plasma components are washed away and a detection mixture containing Glu-plasminogen and a chromogenic plasmin substrate is incubated on the plate. The bound u-PA activates Glu-plasminogen to yield plasmin. The reaction between plasmin and the chromogenic plasmin substrate releases a colored product whose concentration is proportional to the amount of active u-PA in the test sample. After photometrically measuring this reaction, the microtiter plate is washed to remove the activity substrate solution. The u-PA antigen remains bound to the plate. A horseradish peroxidase (POX) conjugated anti-u-PA antibody which recognizes both active u-PA and inactive u-PA is then incubated on the plate. Following incubation and washing of the plate, a POX substrate is used to produce a colored reaction product whose concentration is proportional to the total u-PA content of the test sample.
Human urokinase (u-PA) is an enzyme which functions as an activator of the fibrinolytic enzyme system. Its ability to lyse fibrin clots makes it useful as an effective thrombolytic agent in the research of a variety of clinical disease states including pulmonary embolism and localized thrombosis. Urokinase is synthesized by and released from numerous cell types including endothelial cells, kidney cells and macrophages. Several malignant tumors, especially those of the urogenital and gastrointestinal tracts, have been shown to produce increased quantities of urokinase.
Urokinase exists in three major forms: enzymatically inactive single chain urokinase or pro-urokinase (scu-PA), enzymatically active two chain urokinase (tcu-PA) and urokinase-inhibitor complexes. scu-PA circulates in plasma at a concentration of 1-2 ng/ml and is converted to tcu-PA in vivo by the action of plasmin and kallikrein. Each form of u-PA displays a different activity, different affinities for Glu-plasminogen, and different rates of inhibition by plasma protease inhibitors.
For research administration, pro-urokinase is generally preferred over other forms of this plasminogen activator. Pro-urokinase is not inhibited by plasminogen activator inhibitors and, although no form of urokinase is highly fibrin specific, pro-urokinase is more fibrin-oriented than other forms of u-PA. During thrombolytic testing, pro-urokinase is converted to the active two chain urokinase and is then susceptible to inhibition. The Actibind® u-PA assay employs specific reagents which allow for the measurement of urokinase activity originating from both the active two chain form and the single chain form of the molecule. It does not detect inactive urokinase-inhibitor complexes. u-PA antigen, however, is detected in all three forms (scu-PA, tcu-PA and u-PA inhibitor complexes) by this assay system. This allows the quantification of urokinase inhibitors.
If you wish to differentiate between pro-urokinase and two-chain urokinase, the Technozym scu-PA kit TC11110 determines only single chain urokinase.