The ImmunoDiagnostics PAI-1 Immunoassay is a 3 Hour Solid Phase ELISA designed to measure PAI-1 antigen levels in human serum or plasma samples
The ImmunoDiagnostics Plasminogen Activator Inhibitor-1 (ELISA) is a reliable and reproducible tool for the quantitative detection of PAI-1 production. The assay is intended to use on human serum or plasma.
Numerous application fields exist, but particularly:
- Metabolic Syndrome: PAI-1 has been implicated in the development of adipose tissue as well as the control of insulin signaling in adipocytes
- Fibrinolysis: As a serine protease inhibitor (serpin) that acts as a rapid inhibitor of the plasminogen activators, tPA and uPA, PAI-1 is tightly involved in fibrinolysis and elevated expression of PAI-1 is considered a risk factor for thrombosis.
PAI-1 Antibody Coated Microstrips: One microplate, 12 strips with 8 wells each, 96 dry wells in total. The wells are coated with a mouse monoclonal anti- human PAI-1 antibody. The microplate is sealed in a foil bag. Any unused strips should be returned to the bag and resealed for future use.
Detection Antibody Solution: Concentrate (100x conc). One vial containing 0.12 mL of a biotin conjugated polyclonal anti- human PAI-1. Detection antibody should be diluted with only the 1X Assay Buffer needed at time of use.
Assay Buffer: Concentrate (5x conc). One vial containing 20 mL of buffer for dilution of the detection antibody. If precipitates are observed in the concentrated buffer, warm at 37oC until the precipitates disappear. Dilute 20 mL of 5x Assay Buffer with 80 mL diH2O to make a 1x concentration prior to use.
Human PAI-1 Standard: 2 ng of recombinant human PAI-1 in a buffered protein base, lyophilized. Reconstitute with 1 mL 1x Assay Buffer to prepare 25 ng/mL standard stock. Prepare a serial dilution series with 1x Assay Buffer. The reconstituted standard stock can be aliquoted and stored at -20oC for one month. Avoid repeated freeze/thaw cycles.
Wash Buffer: Concentrate (10x conc). One vial containing 50 mL of 10x wash buffer. If precipitates are observed in the 10x solution, warm at 37oC until the precipitates disappear. Prepare 1x Wash Buffer my mixing 50 mL of 10x Wash Buffer with 450 mL diH2O.
STP-HRP Solution: Concentrate (200x conc). One vial containing 0.06 mL of 200x STP-HRP solution. Briefly spin the 100x vial and dilute the desired amount of 200x STP-HRP solution 1:200 with 1x Assay Buffer to prepare the 1x solution. 100 μL of 1x solution are required per well. Immediately return unused concentrate to 2-8oC storage.
Substrate Solution: One bottle containing 12 mL of TMB (3,3’,5,5’-Tetramethylbenzidine) substrate. Ready-to-use. Do not expose to light.
Stop Solution: One vial containing 12 mL of ready-to-use stop solution.
The PAI-1 ELISA is a quantitative sandwich enzyme immunoassay. Standard, samples, and any desired controls are pipetted into the wells pre-coated with solid-phase capture antibody. After washing away any unbound substances, a biotin-labelled detection antibody, specific for human PAI-1, is added to the wells, and any unbound antibody is washed out. Streptavidin-HRP conjugate is then added and a TMB solution is added to develop color in proportion to the amount of human PAI-1 initially bound. The color development is stopped by addition of an acidifying solution and the optical density is determined. By plotting a standard curve versus measured absorbance, the amount of antigen in the sample can be calculated. The concentration of the antigen is expressed in ng/mL.
The kit contains standards and can be used in any laboratory equipped with a plate reader. The kit is optimized for the quantitative determination of human PAI-1 concentrations in serum and plasma samples. The test can provide results within 3 hours.
Example only! Do not use for calculations
4 Parameter Curve
Log-log Regression Curve
PAI-1 is a single chain glycoprotein that is synthesized in hepatocytes and endothelial cells. In the plasma it is stabilized through interactions with vitronectin or will form inactivation complexes with tPA or uPA. It is these complexes, with tPA and uPA, that allow it to regulate fibrinolysis.
The PAI-1 antigen has been shown to be increased in thrombosis, hepatic disorders, malignant diseases and extract, post-surgery, and during sepsis. Studies show a relationship between PAI-1 concentrations and cardiovascular risk factors (such as obesity, hyperinsulinemia, arteriothrombosis, etc…)