Urokinase Plasminogen Activator Receptor (uPAR) ELISA Kit

Urokinase-type plasminogen activator receptor (uPAR, CD87) is a cell surface receptor that binds urokinase-type plasminogen activator (uPA) with high affinity, thereby facilitating the pericellular activation of plasminogen (see references 1 and 2 for reviews). uPAR, uPA and plasminogen activator inhibitor-1 (PAI-1), form a triad with multiple functions, including regulation of cell attachment, migration, proliferation and differentiation, by both proteolytic and nonproteolytic mechanisms (2). uPAR is anchored to extracellular surfaces through a glycosyl phosphatidylinositol (GPI) linkage, with no transmembrane domain (3). uPAR is synthesized as a 335-amino-acid polypeptide that includes a 22-residue signal peptide (4). A 30-residue peptide is cleaved from the C-terminus during addition of the GPI anchor (3). With loss of the signal peptide and the C-terminal peptide, the mature protein has 283 amino acids. It is variably glycosylated, increasing its mass from about 31kD for the protein backbone to as much as 55kD for the, mature glycoprotein (5). Pro-uPA, a single-chain protein, is activated to a disulfide-linked, two-chain protein by proteolytic cleavage by plasmin (1, 2, 6). Either pro-uPA or the active two-chain uPA bind with high affinity to uPAR. Thus, traces of plasmin activate pro-uPA to uPA, leading to increasing generation of plasmin in a positive feedback loop that is amplified by uPAR. While the initiating event is not clear, the effect is the generation of plasmin at the cell surface, where it degrades the extracellular matrix by activating matrix metalloproteinases. This appears to be a key event in tumor invasiveness and metastasis and in migration of cells in general (1, 2, 7). The functions of the uPA/uPAR system are, however, more extensive than mediation of plasmin formation, and they include non-proteolytic functions (1, 2). uPA/uPAR is localized to focal contact points of cells within the substratum. These sites include intracellular vinculin and the extracellular adhesion molecule vitronectin, suggesting direct adhesive functions and intracellular signalling functions for uPAR. uPAR binds to integrins, and it contains chemotactic activity in its single protease-sensitive region. uPAR has been measured in human plasma (7-9). Soluble uPAR is generated by removal of the GPI anchor by an endogenous phospholipase D, freeing uPAR of its surface attachment 10). uPAR is elevated in plasma of patients with paroxysmal nocturnal hemoglobinuria (7, 8), a manifestation of an inability to add GPI anchors to proteins. It has been postulated that there also may be soluble uPAR due to alternative splicing of the primary transcript (1), as has been demonstrated for mouse uPAR (11). uPAR has been identified in urine, where the level is a consistent fraction of creatinine concentration (12). , The uPAR Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human uPAR in cell culture supernates, serum, plasma, and urine. It contains NS0-expressed recombinant human uPAR and antibodies raised against the recombinant factor. It has been shown to accurately quantitate the recombinant factor. Results obtained using natural human uPAR showed linear curves that were parallel to the standard curves obtained using the kit standards. These results indicate that the uPAR kit can be used to determine relative mass values of natural uPAR. Principle: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for uPAR has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any uPAR present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for uPAR is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of uPAR bound in the initial step. The color development is stopped and the intensity of the color is measured. Kit Components: uPAR Microplate, 1x96 wells , uPAR Conjugate, 1x21ml , uPAR Standard, 1x40ng of recombinant human uPAR , Assay Diluent, 1x11ml , Calibrator Diluent, 1x21ml , Wash Buffer Concentrate (25X), 1x21ml , Color Reagent A, 1x12.5ml of stabilized hydrogen peroxide., Color Reagent B, 1x12.5ml (tetramethylbenzidine)., Stop Solution, 1x6ml (2N sulfuric acid). Storage and Stability: Store components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.