uPA Chromogenic Activity Assay Kit (Indirect Assay)

Catalog No. ABIN612651

Product Details

Target: PLAU (Plasminogen Activator, Urokinase (PLAU))

Reactivity: Human

Detection Method: Colorimetric

Minimum Detection Limit: 0.006 IU/mL

Application: Activity Assay (AcA)

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

Sample Type: Plasma

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

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. Assay Diluent: 30 ml uPA Standard: 1 vial human high molecular weight uPA (1200 IU) Human Plasminogen: 1 vial Plasmin Substrate: 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: 20 μL

Protocol: The assay measures the ability of uPA to activate the plasminogen to plasmin in coupled or indirect assays that contain uPA, plasminogen, and a plasmin-specific synthetic substrate. The amount of plasmin produced is quantitated using a highly specific plasmin substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA in the reaction solution at 405 nm is directly proportional to the uPA enzymatic activity.

Reagent Preparation:

Plasminogen: Add 1.2 ml Assay Diluent. Plasmin Substrate: Add 1.1 ml reagent grader water. Standard Curve: Reconstitute the uPA Standard with 3 ml of Assay Diluent to generate a stock solution of 400 IU/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Dilute the stock solution 1:20 to generate a working solution of 20 IU/ml. For high level of uPA activity samples, prepare triplicate standard points by serially diluting the standard working solution (20 IU/ml) twofold with equal volume of Assay Diluent to produce 10, 5, 2.5, 1.25 and 0.625 IU/ml. For low level of uPA activity samples, dilute the working solution (20 IU/ml) 1:100 with Assay Diluent to yield a solution of 0.2 IU/ml. Prepare triplicate standard points by serially diluting the standard solution (0.2 IU/ml) twofold with equal volume of Assay Diluent to produce 0.1, 0.05, 0.025, 0.0125, and 0.00625 IU/ml. Assay Diluent serves as the zero standard (0 IU/ml). 2 Standard curve for high level of uPA activity samples: Standard Dilution [uPA] (IU/ml) Point P1 1 part Standard (20 IU/ml) 20.000 P2 1 part P1 + 1 part Assay Diluent 10.000 P3 1 part P2 + 1 part Assay Diluent 5.000 P4 1 part P3 + 1 part Assay Diluent 2.500 P5 1 part P4 + 1 part Assay Diluent 1.250 P6 1 part P5 + 1 part Assay Diluent 0.625 P7 Assay Diluent 0.000 Standard curve for low level of uPA activity samples: Standard Dilution [uPA] (IU/ml) Point P1 1 part Standard (0.2 IU/ml) 0.2000 P2 1 part P1 + 1 part Assay Diluent 0.1000 P3 1 part P2 + 1 part Assay Diluent 0.0500 P4 1 part P3 + 1 part Assay Diluent 0.0250 P5 1 part P4 + 1 part Assay Diluent 0.0130 P6 1 part P5 + 1 part Assay Diluent 0.0063 P7 Assay Diluent 0.0000

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.11 M sodium citrate as an anticoagulant. Centrifuge samples at 2,000 x g for 10 minutes and assay.

Assay Procedure:

Add 50 µL of the Assay Diluent to each well of the 96-well plate. Add 10 µL of the Plasminogen to each well. Add 20 µL of uPA Standards or testing samples per well and mix gently. Add 20 µL of Plasmin Substrate to each well and mix gently. Read the absorbance at 405 nm at zero minutes for background O.D. Seal the plate with sealing tape. Incubate the plate at 370C in a humid incubator to avoid drying the plate. For HIGH uPA activity samples, read the absorbances at 405 nm periodically every 5 minutes up to 30 minutes. For LOW uPA activity samples, start to read the absorbances at 405 nm from 3 hours up to 8 hours. 50 µL Assay Diluent 10 µL Plasminogen 20 µL uPA or Samples 20 µL Plasmin Substrate High uPA activity Samples: 370C, read the absorbances at 405 nm every 5 minutes for 30 minutes Low uPA Activity samples: 370C, read the absorbances at 405 nm every hour from 3 hours to 8 hours

Calculation of Results:

Calculate the mean value of the triplicate for each standard and sample. To generate a Standard Curve from the initial reaction time, plot the graph using the standard concentrations on the x-axis and the corresponding mean 405 nm absorbance or change in absorbance per minute (A/min) 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 unopened kit at 2-8°C up to expiration date. Opened reagents may be stored for up to 1 month at 2-8°C. Store reconstituted standard and reagents at -20°C or below.

Target Details

Target: PLAU (Plasminogen Activator, Urokinase (PLAU))

Alternative Name: uPA (PLAU Products)

Synonyms: PLAU Kit, u-PA Kit, ATF Kit, BDPLT5 Kit, QPD Kit, UPA Kit, URK Kit, uPA Kit, UPAM Kit, plasminogen activator, urokinase Kit, urokinase-type plasminogen activator Kit, PLAU Kit, CpipJ_CPIJ002131 Kit, CpipJ_CPIJ006543 Kit, CpipJ_CPIJ013396 Kit, CpipJ_CPIJ013623 Kit, Plau Kit

Background: Urokinase-type plasminogen activator (uPA) is a highly restricted serine protease that converts the zymogen plasminogen to active plasmin, a broad-spectrum serine proteinase capable of degrading most of the major protein components of the extracellular matrix. Binding of uPA to its receptor and subsequent uPA mediated pericellular proteolysis are involved in many process including cell migration and tissue remodeling in angiogeenesis, atherogenesis, tumor cell metastasis, and ovulation. High level of uPA is a poor prognostic marker for aggressive breast cancer, aggressive prostate cancer, bladder cancer and gastric cancer.