DiaPharma Group, Inc.

For Research Use Only.

The scu-PA Reagent Kit is a single chain urokinase antigen ELISA enzyme immunoassay for the determination of scu-PA in human plasma.

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 management 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 1-2ng/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 therapeutic 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, prourokinase is more fibrin-oriented than other forms of u-PA. During thrombolytic therapy, pro-urokinase is converted to the active two chain urokinaseand is then susceptible to inhibition.

Application

The scu-PA Elisa can be used to detect elevated scu-PA antigen levels which may be associated with certain disease states such as tumours of the gastrointestinal or urogenital tracts.

Test Principle

The TC scu-PA test is a solid phase enzyme immunoassay in which a specific anti-u-PA monoclonal antibody coated onto a plastic microtitreplate is used to bind scu-PA and u-PA contained in plasma samples. Following the binding of the antigen to the antibody coated plate, other plasma components are washed away and an enzyme-labelled (POX =horseradish peroxidase) antibody specific for scu-PA only is incubated on the plate. The quantity of POX antibody which binds is proportional to the quantity of scu-PA contained in test samples. Unbound POX antibody is washed away and a substrate which react with the peroxidase enzyme is added, leading to a colour change proportional to the amount of enzyme
bound.

The absorbances of the wells are measured and the values obtained are used to construct a standard curve from which patients' sample values can be extrapolated.

Specificity

The TC scu-PA ELISA measures single chain urokinase only and not double chain urokinase. It is unaffected by and does not recognize other plasminogen activators. This test system measures scu-PA quantities from 0-10 ng/mL.The normal scu-PA value in plasma is 1.8 + 0.6 ng/mL.

Kit Components & Storage

The enclosed reagents are sufficient for five 96- well microtitre plates.

1. One small vial containing a lyophilized monoclonal anti-u-PA antibody.
2. Five bottles containing peroxidase conjugated monoclonal anti-scu-PA antibody (concentrated).
3. Five small vials containing a lyophilized scu- PA standard.

Store all components at 4°C.