PAI-1 tPA Complex ELISA Kit

Catalog No. ABIN612751

Product Details

Target: PAI-1 tPA Complex

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 200 pg/mL

Application: ELISA

Purpose: The AssayMax Human PAI-1/tPA ELISA kit is designed for detection of PAI-1/tPA in human plasma, tissue extracts or cell culture supernatants

Brand: AssayMax

Sample Type: Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Specificity: Reference Values: Normal human plasma PAI-1/tPA concentration has been reported ranging approximately from 1.2 to 4.4 ng/ml (5), and from 2.4 to 8.8 ng/ml (8).

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

Components: PAI-1/tPA Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against PAI-1. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. 1 PAI-1/tPA Standard: Human PAI-1/native human tPA in a buffered protein base (16 ng, lyophilized). Biotinylated tPA Antibody (100x): A 100-fold biotinylated polyclonal antibody against human tPA (80µl). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water

Application Details

Sample Volume: 50 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures PAI-1/tPA in 4 hours. A polyclonal antibody specific for PAI-1 has been pre-coated onto a microplate. PAI-1/tPA complex in standards and samples is sandwiched by the immobilized antibody against PAI-1 and a biotinylated polyclonal antibody against tPA, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 16 ng of human PAI-1/tPA Standard with 1 ml of EIA Diluent to generate a 16 ng/ml of stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the PAI-1/tPA standard solution (16 ng/ml) twofold with equal volume of EIA 2 Diluent to produce 8, 4, 2, 1, 0.5, and 0.25 ng/ml. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [PAI-1/tPA] (ng/ml) P1 1 part Standard (16 ng/ml) 16.000 P2 1 part P1 + 1 part EIA Diluent 8.000 P3 1 part P2 + 1 part EIA Diluent 4.000 P4 1 part P3 + 1 part EIA Diluent 2.000 P5 1 part P4 + 1 part EIA Diluent 1.000 P6 1 part P5 + 1 part EIA Diluent 0.500 P7 1 part P6 + 1 part EIA Diluent 0.250 P8 EIA Diluent 0.000 Biotinylated tPA Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute Wash Buffer Conc. 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. 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 2000 x g for 10 minutes and assay. Dilute samples 1:4 with EIA Diluent. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as anticoagulant). Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Tissue: Extract tissue samples with 100 mM phosphate-buffered saline (pH7.4) containing 1% Triton x-100 and centrifuge at 14000 x g for 20 min. Collect the supernatant and measure the protein concentration. Dilute the tissue extract 1:4 into EIA Diluent and assay. Freeze the remaining extract at -20°C or below.

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 (20 - 30 °C). 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 50 µL of standard or samples per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated tPA Antibody Conjugate to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Wash five times with 200 µL of Wash Buffer. Turn on the microplate reader and set up the program. Add 50 µL of Chromogen Substrate per well and incubate for approximately 12 minutes or till the optimal color density develop. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings. 3

Calculation of Results:

Calculate the mean value of the duplicate or 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 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.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 5.0 % and 7.0% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: The kit should not be used beyond the expiration date.

Storage: 4 °C/-20 °C

Storage Comment: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted 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.

References

Kheirandish-Gozal, Gileles-Hillel, Alonso-Álvarez, Peris, Bhattacharjee, Terán-Santos, Duran-Cantolla, Gozal: "Effects of adenotonsillectomy on plasma inflammatory biomarkers in obese children with obstructive sleep apnea: A community-based study." in: International journal of obesity (2005), Vol. 39, Issue 7, pp. 1094-100, (2015) (PubMed).

Ostrowski, Haase, Müller, Møller, Pott, Perner, Johansson: "Association between biomarkers of endothelial injury and hypocoagulability in patients with severe sepsis: a prospective study." in: Critical care, Vol. 19, pp. 191, (2015) (PubMed).

Elsayh, Mohammed, Zahran, Saad: "Leukocytes apoptosis and adipocytokines in children with beta thalassemia major." in: Clinical and experimental medicine, (2015) (PubMed).

Tazawa, Sato, Tsutiya, Tokeshi, Ohtani-Kaneko: "A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress." in: Thrombosis research, Vol. 136, Issue 2, pp. 328-34, (2015) (PubMed).

Target Details

Target: PAI-1 tPA Complex

Alternative Name: PAI-1/tPA

Background: Type I plasminogen activator inhibitor (PAI-1) is a 50 kDa serpin family member that inhibits tissue- and urokinase-type plaminogen activators (t-PA, u-PA). Whereas tPA is a 68 kDa serine protease that converts the plasminogen into plasmin and facilitates the digestion of fibrin clots. In plasma, half or more of PAI-1 and most tPA present in the circulation is in an inhibited complex. In the resting state in healthy individuals, typically less than 20% of tPA is present in its free form in the plasma. In normal individuals as well as in patients with recurrent venous thrombosis, high PAI-1 plasma concentration is usually associated with high tPA antigen (but not with free tPA) levels. PAI-1/tPA complex, a novel fibrinolytic marker, increases during the pregnancy-associated hypercoagulable state, atherosclerosis and vascular spasm. Determination of PAI-1/tPA complex may provide valuable prognostic information with respect to breast cancer patients and myocardial infarction in patients with manifest coronary heart disease.