Thrombin-Heparin Cofactor II Complex:
The activation of coagulation ultimately leads to the activation of prothrombin to the enzyme thrombin. Unless regulated, thrombin will act on its natural substrates that include fibrinogen, factor V, factor VIII, factor XIII, Protein C, TAFI as well as specific receptors on platelets and endothelial cells. The activity of thrombin in plasma is regulated in part through interaction with protease inhibitors. Based on kinetic rates and physiological concentrations, the primary inhibitor of thrombin in plasma is antithrombin (ATIII), followed by heparin cofactor II (HCII) and a2macroglobulin. The thrombin-heparin cofactor II complex (T-HCII) results when thrombin cleaves a scissile bond near the C-terminus of HCII, forming a covalent, 1:1 acyl enzyme intermediate with HCII with an apparent mass of 102 kD. Calcium is not required for this interaction, but the rate of thrombin inhibition by HCII can be accelerated 1000-fold by optimal concentrations of heparin. Unlike Antithrombin, thrombin inhibition by HCII is also enhanced by dermatan sulphate. T-HCII complexes are cleared from circulation by serpin-enzyme complex receptors on the surface of hepatocytes, with a half-life of 10 minutes (1-3).
Principle of Sandwich-style ELISA:
Affinity-purified antibody to human thrombin is coated onto the wells of a microtiter plate. Any remaining binding sites on the plastic wells are blocked with bovine serum albumin. The plates are washed and plasma or other fluids are applied. The coated antibody will capture the thrombin and thrombin-inhibitor complexes in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to HCII is added to the plate to bind to the captured THCII complexes. 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 THCII complex present in the sample.
Sufficient reagent for 5x96 well plates.
1) Capture Antibody, 1x500ul, affinity purified Sheep antibody to Thrombin for coating plates.
2) Detecting Antibody, 1x500ul, Peroxidase conjugated, affinity purified goat antibody to Heparin Cofactor II (HCII) for detection of HCII complexed to Thrombin.
Storage and Stability:
Store components at -20 degrees C. Stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.