FXII is a serine protease and plays a role in blood coagulation, fibrinolysis, kinin and complement systems. The protein is the zymogen of the serine protease factor XIIa (FXIIa). FXII is converted to FXIIa through autoactivation induced by contact to charged surfaces, also known as the plasma contact system. FXII is predominantly synthesized in the liver and is composed of fibronectin type I and II domains, two epidermal growth factor-like domains, a kringle region, a proline-rich domain and a catalytic domain. Its molecular weight is approximately 80kDa on SDS-PAGE gel electroforeses. The protein circulates in the plasma at a concentration of 30-35 μg/ml.
FXII forms the plasma contact system together with high molecular weight kininogen and plasma kallikrein. FXII autoactivates when these three proteins form a complex on negatively charged nonphysiological surfaces, like inorganic surfaces (eg silicon tubes) or macromolecular organic surfaces (eg heparin) bound to the surface of different cell types, including endothelial cells, platelets and neutrophils. It can trigger blood coagulation and generation of proinflammatory bradykinin. After surface complexation, FXII autoactivates into FXIIa, also called factor XII fragment(XIIf). Once small amounts of kallikrein are formed a positive feedback loop is active leading to enhanced conversion into FXIIa. The activation leads to a series of active enzyme formation. FXIIa converts prekallikrein to kallikrein and kallikrein digests kinogen to liberate proinflammatory bradykinin. Bradykinin triggers inflammatory reactions via activating endothelial cells resulting in vasodilatation, increased vascular permeability and production of other mediators like nitric oxide.
The contact system has the ability to activate the complement system via the classical pathway. Simultaneous activation of both systems may lead to pathological conditions, like hereditary angioedema in individuals with dysfunctional C1-inhibitor (C1-IHB). FXIIa can activate complement protein C1r and to a lesser degree C1s in absence of C1-IHB. This leads to unimpeded bradykinin formation resulting in angioedema. Other interactions with complement system are found on the level of gC1qR and MASP-1.
The human coagulation factor XII ELISA kit is to be used for the in vitro quantitative determination of coagulation factor XII in plasma and serum.
The coagulation factor XII ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours. The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay. Samples and standards are incubated in microtiter wells coated with antibodies recognizing coagulation factor XII. Biotinylated tracer antibody will bind to the captured. Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody. Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB). The enzyme reaction is stopped by the addition of oxalic acid. The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the coagulation factor XII standards (log). The coagulation factor XII concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Linear recovery was determined by performing dilutions with citrate plasma ranging from 500x to 8,000x. This experiment demonstrated that there was linearity with a CV of 3.6%.
Recovery was determined by mixing plasma samples with a known coagulation factor XII concentration in different ratios and measuring them using ELISA. Recovery for these samples ranged between 88% and 97%.
Automated ELISA performance
In case plate washer is used, please note: use of a plate washer can result in higher background and decrease in sensitivity. We advise validation of the plate washer with the manual procedure.
Make sure the plate washer is used as specified for the manual method.