Tissue-type plasminogen activator (tPA) is one of two major physiologic activators of plasminogen in plasma. It is a serine protease of 68 kDa produced primarily in endothelial cells but is also present in monocytes and megakaryocytes. Normal plasma tPA antigen concentrations have been reported from 20ng/ml to 5ug/ml, depending on the assay used, but typically most of the tPA (>90%) is in complex with it’s primary inhibitor, plasminogen activator inhibitor-1 (PAI-1). Structurally, tPA is a single-chain enzyme that consists of a catalytic domain followed by two kringle structures, an EGF domain and a finger domain. The activation of plasminogen by tPA is dependent on the presence of a fibrin cofactor. The binding of both tPA and plasminogen to fibrin is mediated in part through lysine binding sites within the kringle structures of both enzyme and substrate, but also through the finger domain of tPA. Activation of plasminogen by tPA occurs by cleavage after residue Arg560 to produce the two-chain active serine protease plasmin. The activity of tPA is regulated in part by a very short half life in circulation (t1⁄2 of ~4 minutes) and by circulating protease inhibitors PAI-1 and to a lesser extent alpha2macroglobulin.
Principle:Affinity-purified antibody to tPA is coated onto the wells of a microtitre plate. Any remaining binding sites on the plastic wells are blocked with an excess of bovine serum albumin. The plates are washed and plasma or other fluids containing tPA are applied. The coated antibody will capture the tPA in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to tPA is added to the plate to bind to the captured tPA. After washing the plate to remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with o-phenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition of H2SO4 and the color produced is quantified using a microplate reader. The color generated is proportional to the concentration of tPA present in the sample.
Quantity:Sufficient reagents for 5x96 well plates
Kit Components:P4256-33A: Capture antibody, 1x500ul (Yellow cap). Polyclonal affinity purified anti-tPA, 50% glycerol. Suitable for use as coating antibody P4256-33B: Detection antibody 1x500ul (Red cap). Polyclonal affinity purified anti-tPA, 50% glycerol. Suitable for use as detection antibody
Storage and Stability:Store components at -20°C. Stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
