uPA assay kit

Rat uPA activity ELISA assay

Cat. Num. AAA480481

• Synonyms: uPA; Rat uPA activity ELISA assay; Rat uPA Active Antigen ELISA; uPA assay kit

• Procedural Notes: 1. Use aseptic technique when opening and dispensing reagents.; 2. This kit is designed to work properly as provided and instructed. Additions, deletions, or substitutions to the procedure or reagents are not recommended, as they may be detrimental to the assay.; 3. Exercise universal precautions during the performance or handling of this kit or any component contained therein.
• Sample Collection and Preparation: Collect 9 volumes of blood in 1 volume of 0.1 M trisodium citrate or acidified citrate, preferably using Stabilyte evacuated vials. Immediately after collection of blood, samples must be centrifuged at 3000 x g for 15 minutes. It is important to ensure a platelet free preparation as platelets can release PAI-1, which in turn could potentially form a complex with uPA. The plasma must be transferred to a clean plastic tube and must be stored on ice prior to analysis. The uPA activity samples collected in the Stabilyte media are stable for up to 24 hours or stored at -20 degree C for up to one month and thawed three times without loss of uPA activity.
• Reagent Preparation: 1. 10x Wash Buffer: Dilute the 50 mL of concentrate to 1x with 450 mL of DI water prior to use.; 2. TBS Buffer: 0.1 M Tris, 0.15 M NaCl, pH 7.; 4.; 3. 3% BSA Blocking Buffer: 3% BSA in TBS Buffer.; 4. Biotinylated PAI-1: Reconstitute with 3% BSA Blocking Buffer as directed on the vial; and vortex; gently to mix. Prepare immediately prior to use.; 5. Primary Antibody: Reconstitute with 3% BSA Blocking Buffer as directed on the vial; and vortex; gently to mix. Prepare immediately prior to use.; 6. Secondary Antibody: Dilute with 3% BSA Blocking Buffer as directed on the vial and; vortex gently; to mix. Prepare immediately prior to use.
• Standard Preparation: Reconstitute the Standard as directed on the vial to give a 100 ng/mL Standard Stock Solution. Do not prepare the standards until you are ready to apply them to the plate.; Table 1: Preparation of Standard Curve; Standard; uPA; Concentration; (ng/mL); Blocking; Buffer; (muL); Transfer; Volume; (muL); Transfer; Source; Final; Volume; (muL); S9 10 900 100 Stock Vial 500; S8 5 500 500 S9 600; S7 2 600 400 S8 500; S6 1 500 500 S7 500; S5 0.5 500 500 S6 500; S4 0.25 500 500 S5 600; S3 0.1 600 400 S4 500; S2 0.05 500 500 S3 500; S1 0.025 500 500 S2 1,000; B0 0 500 --- --- 500
• Preparation and Storage: 1. Store this kit and its components at 4 degree C until use.; 2. The reconstituted Standard and Primary Antibody may be stored at -70C for later use.; DO NOT; freeze/thaw the Standard or Primary Antibody more than once.

Testing Data

Testing Data #2

Related Product Information for uPA assay kit

Introduction: This rat urokinase plasminogen activator activity assay is for the quantitative determination of active plasminogen activator in rat plasma. Urokinase plasminogen activator (uPA) is a serine that activates plasminogen to plasmin in the blood fibrinolytic system. It is also implicated in events related to cell invasion/migration (3).

Principle of assay: Functionally active uPA will bind to the biotinylated human PAI-1 coated on the microtiter plate. Only free active enzyme will react with the PAI-1 on the plate. Inactive or complexed enzyme will not be detected. After appropriate washing steps, monoclonal mouse anti-rat uPA primary antibody binds to the captured enzyme. Excess antibody is washed away, and bound monoclonal antibody is then reacted with the secondary antibody conjugated to horseradish peroxidase. TMB substrate is used for color development at 450 nm. A standard calibration curve is prepared along with the samples to be measured using dilutions of uPA.

References

1. Declerck, P.J., et al.; (1995) Thromb Haemostas: 74(5): 1305-9
2. Singh, I., et al.; (2003) Circulation: 107(6): 869-875
3. Kjøller, L,; (2002) Biol. Chem.: 383: 5-19
4. Yang, Y.H., et al.; (2001) J. Immunol.: 167(2): 1047-52