Factor x (Fx) Chromogenic Activity Assay Kit

Catalog No. ABIN612645

Product Details

Target: Coagulation Factor X (F10)

Reactivity: Human

Detection Method: Colorimetric

Minimum Detection Limit: 30 U/mL

Application: Activity Assay (AcA)

Purpose: The AssaySense Human Fx Chromogenic Activity Assay Kit is developed to determine human Fx activity in plasma and cell culture

Sample Type: Plasma, Cell Culture Supernatant

Components: The activity assay kit contains sufficient reagents to perform 100 tests using microplate method. Microplate: One 96-well polystyrene microplate (12 strips of 8 wells) Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Sample Diluent (6x): A six-fold concentrate (10 ml) Human Fx Standard: 1 vial (10 µg) Assay Diluent: A working solution (10 ml) RVV: 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)

Application Details

Sample Volume: 20 μL

Protocol: The assay measures the activation of zymogen Fx to Fxa by RVV. The amidolytic activity of the Fxa is quantitated 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 Fx enzymatic activity.

Reagent Preparation:

Sample Diluent: Dilute Sample Diluent Concentrate 1:6 with reagent grade water. Standard Curve: Reconstitute the Fx Standard (10 g) with 1.25 ml of Sample Diluent to generate a stock solution of 8 g/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. For high level of Fx standard, prepare duplicate or triplicate standard points by serially diluting the standard solution with equal volume of Sample Diluent to produce 4, 2, 1, 0.5, 0.25 and 0.125 g/ml solutions. Sample Diluent serves as the zero standard ( g/ml). Any remaining solution should be frozen at -20°C. For low level of Fx standard, dilute stock solution (8 g/ml) 1:8 with Sample Diluent to produce 1 g/ml standard. Prepare duplicate or triplicate standard points by serially diluting the standard solution 1:4 with equal volume of Sample Diluent to produce 0.25, 0.0625 and 0.0156 g/ml solutions. Sample Diluent serves as the zero standard ( g/ml). Any remaining solution should be frozen at -20°C. Standard curve for high level of Fx activity samples: Standard Point Dilution [Fx] ( g/ml) 1 part Standard (8 g/ml) P1 8.000 P2 1 part P1 + 1 part Sample Diluent 4.000 P3 1 part P2 + 1 part Sample Diluent 2.000 P4 1 part P3 + 1 part Sample Diluent 1.000 P5 1 part P4 + 1 part Sample Diluent 0.500 P6 1 part P5 + 1 part Sample Diluent 0.250 P7 1 part P6 + 1 part Sample Diluent 0.125 P8 Sample Diluent 0.000 2 Standard curve for low level of Fx activity samples: Standard Point Dilution [Fx] ( g/ml) P1 1 part P1 + 7 parts Sample Diluent 1.000 P2 1 part P1 + 3 parts Sample Diluent 0.250 P3 1 part P2 + 3 parts Sample Diluent 0.0625 P4 1 part P3 + 3 parts Sample Diluent 0.0156 P5 Sample Diluent 0.000 RVV: Add 1.2 ml of Sample Diluent. Allow the RVV to sit for 10 minutes to dissolve. Fxa Substrate: Add 1.1 ml of reagent grade water. Allow the substrate to sit for 10 minutes to dissolve.

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 1:10 with Sample Diluent and assay immediately. Samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 15 minutes at 40C to remove debris. Collect supernatants and assay.

Assay Procedure:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at 37 0C for chromogenic activity assay. Seal the plate with sealing tape at each step. Remove excess microplate strips from the plate frame. Add 20 µL of Factor x Standard or diluted sample to each well. Assay Mix: At room temperature, freshly prepare the desired volume of the Mix by combining the following reagents according to the assay numbers (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 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

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.

Target Details

Target: Coagulation Factor X (F10)

Abstract: F10 Products

Synonyms: FX Kit, FXA Kit, Cf10 Kit, fX Kit, fi12c10 Kit, wu:fi12c10 Kit, F10 Kit, f10 Kit, coagulation factor X Kit, Coagulation factor X Kit, coagulation factor 10 L homeolog Kit, F10 Kit, f10 Kit, CpipJ_CPIJ012712 Kit, CpipJ_CPIJ014863 Kit, CpipJ_CPIJ016937 Kit, CpipJ_CPIJ017791 Kit, fa10 Kit, f10.L Kit

Background: Factor x (Fx) is a plasma serine protease zymogen involved in the blood coagulation cascade. Fx is purified from plasma as a two-chain protein consisting of a 45-kDa heavy chain and a 17-kDa light chain. The Fx heavy chain is cleaved during coagulation by several different proteases including the intrinsic xase complex, the Fx-activating enzyme from Russell's viper venom (RVV) and trypsin, and also by extrinsic (tissue factor/factor VIIa) pathway to give an active enzyme Fxa. Fxa as the activator of prothrombin occupies a central position linking the two blood coagulation pathways.