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  • 产品名称：FactorVII(FVII)ChromogenicActivityAssayKit

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FactorVII(FVII)ChromogenicActivityAssayKit
详情介绍：
Purpose The AngioSense Human FVII Chromogenic Activity Assay Kit is developed to determine human FVII activity in plasma and cell culture Supernatants
Sample Type Plasma
Detection Method Colorimetric
Specificity This assay recognizes both natural and recombinant human FVII.
Components The activity assay kit contains sufficient reagents to perform 100 tests using microplate method. FVII Microplate: one 96-well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against human FVII. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human FVII Standard: 1 vial (1.6 IU) EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Assay Diluent: 30 ml rhTF (lipoprotein): 1 vial recombinant human TF lipoprotein. Human Fx: 1 vial Fxa Substrate: 2 vials Storage Condition
Material not included Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)
Alternative Name Factor VII (FVII) (F7 ELISA Kit Abstract)
Background Factor VII (FVII) is a vitamin K-dependent plasma glycoprotein that is synthesized in the liver and circulates in blood as a single-chain inactive zymogen with a molecular mass of 50 kDa. Upon tissue damage and vascular injury, the cell surface receptor and cofactor tissue factor (TF) binds and allosterically activates FVII to its active form, FVIIa. The TF/FVIIa complex catalyzes the conversion of both factor Ix to factor Ixa and factor x to factor xa to initiate coagulation via the extrinsic pathway. Very low levels of FVII are associated with severe coagulation disorders. Elevated plasma levels of FVII coagulant activity constitute an independent risk factor for fatal outcomes of coronary heart disease in middle-aged men.
Sample Volume 100 μL
Plate Pre-coated,Strips (12 x 8)
Protocol The assay couples immunofunctional and indirect amidolytic assay. A monoclonal antibody specific for human FVII has been pre-coated onto a microplate and active FVII is bound to the immobilized antibody. The assay measures the ability of lipoprotein TF/FVIIa to activate factor x (Fx) to factor xa. The amidolytic activity of the TF/FVIIa complex is quantitated by the amount of Fxa produced using a highly specific Fxa substrate releasing a yellow para- nitroaniline (pNA) chromophore. The change in absorbance of the pNA at 405 nm is directly proportional to the FVII enzymatic activity.
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the FVII Standard with 4 ml of EIA Diluent to generate a solution of 0.4 IU/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the standard solution (0.4 IU/ml) 1:4 with EIA Diluent to produce 0.1, 0.025, 0.00625 and 0.00106 IU/ml. EIA Diluent serves as the zero standard (0 IU/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [FVII] (IU/ml) P1 1 part Standard 0.4000 P2 1 part P1 + 3 part EIA Diluent 0.1000 P3 1 part P2 + 3 part EIA Diluent 0.0250 P4 1 part P3 + 3 part EIA Diluent 0.0063 P5 1 part P4 + 3 part EIA Diluent 0.0011 P6 EIA Diluent 0.0000 Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. Any remaining solution should be frozen at -20°C. rhTF: Add 1.2 ml of reagent grade water. Any remaining solution should be frozen at -20°C. Fx: Add 1.2 ml reagent grade water. Any remaining solution should be frozen at -20°C. Fxa Substrate: Add 1.1 ml of reagent grade water. Any remaining solution should be frozen at -20°C.
Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and assay. Samples can use no dilution or 1:16 with EIA Diluent. Store undiluted samples at -20°C or below, for up to 3 months. Avoid repeated freeze- thaw cycles. Cell Culture Supernatants: Collect cell culture media and centrifuge at 3000 x g for 10 minutes at 40C to remove debris.
Assay Procedure

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature for specific sample binding and at 37 0C for chromogenic activity assay. Seal the plate with sealing tape at each step. Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely and store in a vacuum desiccator to minimize exposure to water vapor. Add 100 µL of standard or sample per well. Cover wells and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Freshly prepare the desired volume of the Assay Mix by combining the following reagents according to the number of wells in the assay (n) plus one.
Calculation of Results

Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot 4-parameter graph or log-log graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis and draw a best fit curve through the points on the graph. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
Restrictions For Research Use only
Storage -20 °C
Storage Comment Store kit at -20°C upon arrival up to the expiration date. Opened reagents may be stored for up to 1 month at -20°C. Store reconstituted standard and reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Supplier Images
ELISA image for Factor VII (FVII) Chromogenic Activity Assay Kit (ABIN612644) Factor VII (FVII) Chromogenic Activity Assay Kit
Background publications Arbini, Pollak, Bayleran, High, Bauer: "Severe factor VII deficiency due to a mutation disrupting a hepatocyte nuclear factor 4 in the factor VII promoter." in: Blood, Vol. 89, Issue 1, pp. 176-82, 1997 (PubMed).

Junker, Heinrich, Schulte, van de Loo, Assmann: "Coagulation factor VII and the risk of coronary heart disease in healthy men." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 17, Issue 8, pp. 1539-44, 1997 (PubMed).

Bajaj, Rapaport, Brown: "Isolation and characterization of human factor VII. Activation of factor VII by factor Xa." in: The Journal of biological chemistry, Vol. 256, Issue 1, pp. 253-9, 1981 (PubMed).

Davie, Fujikawa, Kurachi, Kisiel: "The role of serine proteases in the blood coagulation cascade." in: Advances in enzymology and related areas of molecular biology, Vol. 48, pp. 277-318, 1979 (PubMed).

Kisiel, Davie: "Isolation and characterization of bovine factor VII." in: Biochemistry, Vol. 14, Issue 22, pp. 4928-34, 1976 (PubMed).