Factor VII (FVII) Chromogenic Activity Assay Kit

Catalog No. ABIN612644

Product Details

Target: Factor VII (F7) (Coagulation Factor VII (F7))

Reactivity: Human

Detection Method: Colorimetric

Minimum Detection Limit: 5.1 mIU/mL

Application: Activity Assay (AcA)

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

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

Components:

• Human Factor VII Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human FVII.
• Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay.
• Human Factor VII Standard: Lyophilized (240 mIU, 84 ng).
• EIA Diluent Concentrate (10x): A 10-fold concentrated buffered proteinbase (20 ml).
• Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml).
• Assay Diluent (1x): Buffered protein base (20 ml).
• rhTF (Lipoprotein): Recombinant human TF lipoprotein (lyophilized).
• Human FX: Lyophilized.
• FXa Substrate: Lyophilized, 2 vials.

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: Assay Summary

1. Add 100 µl of Standard or Sample per well.Incubate 2 hours at room temperature.
2. Wash, then add 80 µl of Assay Mix per well. Incubate 30 minutes at 37°C.
3. Add 20 µl of Factor Xa Substrate per well.Read the absorbance at 405 nm for a zero minute background reading. Cover and incubate at 37°C.
4. Read every 10 minutes for 50 minutes at 405 nm. Cover and incubate at 37°C after each reading.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use.

• EIA Diluent Concentrate (10x): Dilute the EIA Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. When diluting the concentrate, make sure to rinse the bottle thoroughly to extract any precipitates left in the bottle. Mix the 1x solution gently until the crystals have completely dissolved. Store for up to 30 days at 2-8°C.
• Human Factor VII Standard: Reconstitute the Human Factor VII Standard (240 mIU, 84 ng) with 1.2 ml of EIA Diluent to generate a 200 mIU/ml (70 ng/ml) standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (200 mIU/ml) 2-fold with equal volume of EIA Diluent to produce 100, 50, 25, and 12.5 mIU/ml solutions. EIA Diluent serves as the zero standard (0 mIU/ml). Any remaining stock solution should be stored at -20°C and used within 30 days. Avoid repeated freeze-thaw cycles.
• Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. When diluting the concentrate, make sure to rinse the bottle horoughly to extract any precipitates left in the bottle. Mix the 1x solution gently until the crystals have completely dissolved.
• rhTF: Add 1.1 ml of reagent grade water to generate a 1x stock solution. Allow the vial to sit for 10 minutes with gentle gitation prior to use; keep the vial on ice. Any remaining stock solution should be stored at -20°C and used within 30 days. Avoid repeated freeze-thaw cycles.
• Human FX: Add 1.2 ml of reagent grade water to generate a 1x stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to use; keep the vial on ice. Any remaining stock solution should be stored at -20°C and used within 30 days. Avoid repeated freeze-thaw cycles.
• FXa Substrate: Add 1.1 ml of reagent grade water to generate a 1x stock
  solution. Allow the vial to sit for 10 minutes with gentle agitation prior to use; keep the vial on ice. Aliquot remaining stock solution to limit repeated freeze-thaw cycles. This solution should be stored at -20°C and used within 10 days.

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.

Calculation of Results:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve from the optimal reaction time, plot the graph using the standard concentrations on the x-axis and the corresponding mean 405 nm absorbance (OD) or change in absorbance per minute (∆A/min) on the y-axis after subtracting the background. The best fit line can be determined by regression analysis of the 4-parameter curve.
• Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor.

Restrictions: For Research Use only

Handling

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.

Target Details

Target: Factor VII (F7) (Coagulation Factor VII (F7))

Alternative Name: Factor VII (FVII) (F7 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.