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SERPINE1 ELISA Kit (serpin Peptidase Inhibitor, Clade E (Nexin, Plasminogen Activator Inhibitor Type 1), Member 1)

Details for Product SERPINE1 ELISA Kit No. ABIN612750, Supplier: Log in to see
  • PAI
  • PAI-1
  • PAI1
  • PLANH1
  • si:ch211-138a11.1
  • Planh1
  • B230326M24Rik
  • PI-7
  • PI7
  • PN-1
  • Spi4
  • HsT1201
  • PAI-2
  • PAI2
  • PLANH2
  • PAI1A
  • Pai1
  • Pai1aa
  • Planh
  • serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1
  • plasminogen activator inhibitor 1
  • plasminogen activator inhibitor type 1
  • serine (or cysteine) peptidase inhibitor, clade E, member 1
  • serine (or cysteine) peptidase inhibitor, clade E, member 2
  • serpin peptidase inhibitor, clade B (ovalbumin), member 2
  • serpine1
  • CpipJ_CPIJ011718
  • CpipJ_CPIJ016298
  • PAI-1
  • Serpine1
  • Serpine2
Kits with alternative reactivity to:
Methode Type
Sandwich ELISA
Minimum Detection Limit
300 pg/mL
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Supplier Product No.
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Purpose The AssayMax Human PAI-1 ELISA kit is designed for detection of PAI-1 in human plasma, tissue extracts, milk, saliva, and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Specificity This assay recognizes both natural and recombinant human PAI-1. Reference Values: Normal human platelet-poor plasma concentration of PAI-1 has been reported to range from 5 to 40 ng/ml (10). The variability was due in part to the marked diurnal variation on PAI-1, with lower values in the afternoon than in the morning, and also to age-related changes.
Components PAI-1 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. PAI-1 Standard: Human PAI-1 in a buffered protein base (80 ng, lyophilized). Biotinylated PAI-1 Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against PAI-1 (80 l). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). 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 pipettes) Deionized or distilled reagent grade water
Alternative Name Plasminogen Activator Inhibitor-1 (PAI-1) (SERPINE1 ELISA Kit Abstract)
Background Type I plasminogen activator inhibitor (PAI-1) is a 43 kDa serpin family member that inhibits tissue- and urokinase-type plasminogen activators (t-PA, u-PA). This protein appears to be an important regulator of plasminogen activation by t-PA and extracellular proteolysis by u-PA. The plasminogen activator proteolytic enzyme systems are important not only for fibrinolysis but also for extracellular matrix remodeling, and have been implicated in a number of normal and pathological processes including angiogenesis, ovulation and embryogenesis, thrombotic and hemorrhagic disorders, connective tissue diseases, neoplasm and sepsis. PAI-1 is a prognosticator in breast cancer , gastric cancer , various forms of lung cancer and cervical cancer.
Pathways p53 Signaling
Sample Volume 50 μL
Assay Time < 4 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures PAI-1 in less than 4 hours. A polyclonal antibody specific for PAI-1 has been pre-coated onto a microplate. PAI-1 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for PAI-1, 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. MIx Diluent Concentrate (10x): Dilute MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 80 ng of human PAI-1 Standard with 4 ml of MIx Diluent to generate a stock standard solution of 20 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The stock standard solution can be further dilute 1:4 with MIx to generate standard solution of 5 ng/ml. Prepare duplicate or triplicate standard points by serially diluting the PAI-1 standard solution twofold with equal volume of MIx Diluent to produce 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [PAI-1] (ng/ml) P1 Standard (20 ng/ml) + 3 parts MIx Diluent 5.000 P2 1 part P1 + 1 part MIx Diluent 2.500 P3 1 part P2 + 1 part MIx Diluent 1.250 P4 1 part P3 + 1 part MIx Diluent 0.625 P5 1 part P4 + 1 part MIx Diluent 0.313 P6 1 part P5 + 1 part MIx Diluent 0.156 P7 1 part P6 + 1 part MIx Diluent 0.078 P8 MIx Diluent 0.000 Biotinylated PAI-1 Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer 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 MIx 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 4000 x g for 10 minutes to obtain platelet-poor plasma. Dilute sample supernatants 1:16 with MIx Diluent and assay. The undiluted samples can be stored for up to 3 months at -20°C or below -20°C. Avoid repeated freeze-thaw cycles. The time of plasma collection should be standardized as PAI-1 levels show the marked diurnal variation. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. The samples can be stored at -20°C or below. Avoid repeated freeze-thaw cycles. Tissue: Extract tissue samples with 50 mM Tris-buffered saline (pH7.4) containing 0.5% Triton x-100 and centrifuge at 14000 x g for 30 min. Collect the supernatant and measure the protein concentration. Dilute the tissue extract 1:4 into MIx Diluent and assay. The undiluted samples can be stored at -20°C or below. Saliva: Collect saliva using sample tube. Centrifuge samples at 600 x g for 10 minutes. Saliva dilution is suggested at 1:2 in MIx Diluent, however, the user should determine the optimal dilution factor. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 600 x g for 10 minutes. Milk dilution is suggested at 1:2 in MIx Diluent, however, the user should determine the optimal dilution factor. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles. 2
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 to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample 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 manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated PAI-1 Antibody to each well and incubate for one hour. Wash a microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. 3 Add 50 µL of Chromogen Substrate per well and incubate for approximately 15 minutes or till the optimal blue color density develops. Gently tap the 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. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. 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.

Calculation of Results

Calculate the mean value of the 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 dilution factor. 3 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 4.7% and 7.2% respectively.
Restrictions For Research Use only
Handling Advice Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. 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 MIx 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.
Product cited in: Cetinkalp, Felekoglu, Karadeniz, Boyacıoglu, Delen, Yildirim, Yilmaz: "Comparison of the effects of intensive insulin treatment modalities on cardiovascular biomarkers in type 1 diabetes mellitus." in: Diabetes & metabolic syndrome, Vol. 9, Issue 3, pp. 157-62, 2015 (PubMed).

Wang, Long, Wang, Sun: "Association of the plasminogen activator inhibitor-1 (PAI-1) Gene -675 4G/5G and -844 A/G promoter polymorphism with risk of keloid in a Chinese Han population." in: Medical science monitor : international medical journal of experimental and clinical research, Vol. 20, pp. 2069-73, 2014 (PubMed).

Johansson, Sørensen, Perner, Welling, Wanscher, Larsen, Ostrowski: "High sCD40L levels early after trauma are associated with enhanced shock, sympathoadrenal activation, tissue and endothelial damage, coagulopathy and mortality." in: Journal of thrombosis and haemostasis : JTH, Vol. 10, Issue 2, pp. 207-16, 2012 (PubMed).

Vilas, Gomis, Blanco, Cortés, Millán, Ossa, Dorado, López-Cancio, Suñol, Dávalos: "Circadian Rhythms in the Efficacy of Intravenous Alteplase in Patients With Acute Ischemic Stroke and Middle Cerebral Artery Occlusion." in: Chronobiology international, 2012 (PubMed).

Background publications Schneiderman, Loskutoff: "Plasminogen activator inhibitors." in: Trends in cardiovascular medicine, Vol. 1, Issue 3, pp. 99-102, 2011 (PubMed).

Coker, Thomas, Clair, Hendrick, Krombach, Galis, Spinale: "Myocardial matrix metalloproteinase activity and abundance with congestive heart failure." in: The American journal of physiology, Vol. 274, Issue 5 Pt 2, pp. H1516-23, 1998 (PubMed).

Kobayashi, Fujishiro, Terao: "Impact of urokinase-type plasminogen activator and its inhibitor type 1 on prognosis in cervical cancer of the uterus." in: Cancer research, Vol. 54, Issue 24, pp. 6539-48, 1995 (PubMed).

Pedersen, Grøndahl-Hansen, Francis, Osterlind, Hansen, Danø, Brünner: "Urokinase and plasminogen activator inhibitor type 1 in pulmonary adenocarcinoma." in: Cancer research, Vol. 54, Issue 1, pp. 120-3, 1994 (PubMed).

Vassalli, Sappino, Belin: "The plasminogen activator/plasmin system." in: The Journal of clinical investigation, Vol. 88, Issue 4, pp. 1067-72, 1991 (PubMed).

Loskutoff, Sawdey, Mimuro: "Type 1 plasminogen activator inhibitor." in: Progress in hemostasis and thrombosis, Vol. 9, pp. 87-115, 1989 (PubMed).

Booth, Simpson, Croll, Bennett, MacGregor: "Plasminogen activator inhibitor (PAI-1) in plasma and platelets." in: British journal of haematology, Vol. 70, Issue 3, pp. 327-33, 1989 (PubMed).

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