Factor VIIa (FVIIa) Chromogenic Activity Assay Kit

Catalog No. ABIN612646

Product Details

Target: Factor VIIa

Reactivity: Human

Detection Method: Colorimetric

Minimum Detection Limit: 0.007 µg/mL

Application: Activity Assay (AcA)

Purpose: The AngioSense Human FVIIa Chromogenic Activity Assay Kit is developed to determine human FVIIa activity in plasma and cell culture supernatants

Sample Type: Plasma, Cell Culture Supernatant

Specificity: This assay recognizes both natural and recombinant human FVIIa.

Components: The activity assay kit contains sufficient reagents to perform 100 tests using microplate method. FVIIa Microplate: one 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human FVIIa. 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 FVIIa Standard: 1 vial, lyophilized (0.5 µg/ml). 1 Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Assay Diluent: 1x, 30 ml. Human Fx: 1 vial, lyophilized. Fxa Substrate: 2 vials, lyophilized. 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)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The assay couples immunofunctional and indirect amidolytic assay. A polyclonal antibody specific for human FVIIa has been pre-coated onto a microplate and FVIIa is bound to the immobilized antibody. The assay measures the ability of FVIIa to activate factor x (Fx) to factor xa. The amidolytic activity of the FVIIa 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 FVIIa enzymatic activity.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. Standard Curve: Reconstitute the FVIIa Standard with 1 ml of Assay Diluent to generate a stock solution of 0.5 g/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (0.5 g/ml) 1:4 with Assay Diluent to produce 0.125, 0.03125, 0.025, and 0.0078 g/ml. Assay Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [FVIIa] ( g/ml) P1 1 part Standard Stock (0.5 g/ml) 0.500 P2 1 part P1 + 3 parts Assay Diluent 0.125 P3 1 part P2 + 3 parts Assay Diluent 0.03125 P4 1 part P3 + 3 parts Assay Diluent 0.0078 P5 Assay Diluent 0.0000 Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. Any remaining solution should be stored at 2-8°C. Assay Diluent (1x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Any remaining solution should be stored at 2-8°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. Dilute samples 1:2 with Assay 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 and assay. Samples can be stored at

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 Factor VIIa Standard or Sample per well. Cover wells and incubate overnight. 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 triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis of the linear portion of the curve. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. 3 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

Handling

Storage: 4 °C/-20 °C

Storage Comment: Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store FVIIa Standard, Fx, and Fxa Substrate at -20°C Store Microplate, Diluent Concentrate, and Wash Buffer at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Opened Diluent (1x) may be stored for up to 1 month at 2-8°C.

Target Details

Target: Factor VIIa

Alternative Name: Factor VIIa (FVIIa) (Factor VIIa Products)

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.