Description
Intended Uses: This Protein-C ELISA is a solid phase enzyme immunoassay for the determination of Protein-C in citrated human plasma.
Principle of the Assay: This test is using microplates coated with a capture antibody specific for human Protein-C. Diluted plasma samples (1:51) are incubated in the wells allowing Protein C present in the plasma to bind to the antibody. The unbound fraction is removed by washing. Afterwards anti-human Protein- C detection antibody conjugated to horseradish peroxidase (conjugate) is incubated and reacts with the antigen- antibody complex on the microwell surface. Following incubation, unbound conjugate is washed off. Addition of TMB-substrate generates an enzymatic colorimetric (blue) reaction, which is stopped by diluted acid (color changes to yellow). The rate of color formation from the chromogen is measured in optical density units with a spectrophotometer at 450 nm. Using a curve prepared from the Reference Plasma provided with the kit, the Protein C antigen relative percent concentration in the plasma samples can be determined.
Background: Protein C is a vitamin K-dependent inactive zymogen of a serine protease that is mainly synthesized by hepatocytes in the liver. It has a molecular weight of 62 kDa and is present at a concentration of 4 ug/ml in the plasma. Activated Protein C (aPC) is a key component of the Protein C anticoagulant system that is activated by the binding of thrombin to the endothelial transmembrane receptor thrombomodulin. The complex of thrombin and thrombomodulin activates Protein C and the activated Protein C in turn forms a complex with its cofactor Protein S that has a high affinity to phospholipid membranes. This is of physiological importance since aPC inactivates preferentially the membrane-bound coagulation factors Va and VIIIa. Additionally, activated Protein C possesses profibrinolytic activity by inhibiting plasmin activator inhibitor-1 (PAI-1). Protein C deficiency may be inherited or acquired and is associated with a variably increased risk of thrombosis. The prevalence of Protein C deficiency has been estimated to be up to one case per 300 in the general population. Nearly 50-80 % of individuals with inherited Protein C deficiency will experience a thrombotic event before the age of 30-45. Individuals with a homozygous Protein C deficiency may suffer from neonatal purpura fulminans or massive venous thrombosis. Acquired Protein C deficiency is often associated with liver disease, surgery, oral anticoagulant therapy, antiphospholipid syndrome, etc. Protein C deficiency is classified in two states. Type I deficiency is a reduction in the level of Protein C. Type II deficiency is characterized by a reduced Protein C activity, with normal antigen level