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…)
| How does a specific gene polymorphism, PAI-1 4G genotype, relate to CVD risk? |
| For a detailed description of the 4G polymorphism, read Kohler et al. PAI-1 and Coronary Artery Disease. NEJM 2000; 342 (24): 1792-1801. In some studies, the 4G allele (four guanine bases) was significantly associated with high plasma PAI-1 concentrations, and was most strongly associated with previous MI, as well as risk of future MI. Studies have shown that subjects who are homozygous for the 4G allele have plasma PAI-1 concentrations approximately 25% higher than those with the 5G allele (5 guanine bases). Similarly, among patients with hypertriglyceridemia, those with the 4G allele also have higher plasma PAI-1 concentrations than those with the 5G allele. There are still conflicting data on the strength of the relation between PAI-1 gene polymorphism and MI, but it is suggested that the 4G allele is more likely to contribute to MI, particularly in the presence of hypertriglyceridemia. |
| Won’t tPA be inhibited by PAI-1? |
| tPA is inhibited in vitro by plasminogen activator inhibitor, so something must be done to avoid this. Acidification of whole blood is therefore performed immediately after withdrawal. This can be done by mixing 1 ml of the citrated blood with 1 ml acetate buffer. |
| What factors cause increased and decreased levels of t-PA and PAI? |
| There are numerous physiological factors that influence tPA and PAI antigen and activity level. For a complete list, please request the tPA monograph from DiaPharma. One interesting feature of the fibrinolytic system is the circadian variation in tPA and PAI-1level. Free tPA levels are lowest in the morning, increase during the day, and reach their peak activity level in the late afternoon. tPA and PAI-1 antigen are highest in the early morning and decrease during the day. This may help explain the high incidence of MI and stroke in the morning hours. Other factors that influence tPA and PAI-1 include alcohol, drugs, oral contraceptives, exercise, food, heparin administration, pregnancy, smoking, etc. |
| What are some of the inhibitors to t-PA? |
| PAI-1, PAI-2, PAI-3, protease nexin, a2-macroglobulin, trypsin inhibitor, and C1 Inhibitor all inhibit tPA. Plasminogen activator inhibitor 1 (PAI-1) is the most efficient inhibitor of tPA in plasma. It is a serine protease inhibitor (serpin) that acts as a pseudo-substrate for its target protease, with which it forms an inactive complex. PAI-1 is synthesized by several cell types including endothelial cells and hepatocytes and is present in platelets, placenta, and serum. The normal concentration range of PAI-1 in plasma is 5-40 mg/l and the normal activity is 0-20 AU/ml.
PAI-2 is a serpin with a higher affinity for u-PA (urinary-type plasminogen activator, or urokinase) than for t-PA. It is often only detectable during pregnancy, specifically in the third trimester. PAI-3 is also called Protein C inhibitor, and inhibits u-PA and thrombin, and is present in plasma and urine. |
| What is the importance of t-PA and PAI in clinical research? |
| Elevated t-PA and PAI-1 antigen and reduced t-PA activity may be associated with cardiovascular disease. t-PA is given to stroke and heart attack victims shortly after the event to help break up the clot. |
