Technozym® PAI-1 Actibind® ELISA is a high sensitive complete sandwich ELISA for the quantitative determination of active PAI-1 antigen in human plasma. The kit allows determination of active PAI-1 antigen in plasma also with research disseminated intravascular coagulation and in research of atheriosclerotic disease. The Plasminogen Activator Inhibitor type 1 (PAI-1) Actibind ELISA Kit is based on the immobilization of functionally active t-PA to plates by means of a monoclonal antibody. PAI-1 contained in the test samples binds to t-PA and is then quantified using a peroxidase-labeled monoclonal anti-PAI-1 antibody. The assay recognizes PAI-1 active form with no interference from PAI-2 (5 U/mL) and PAI-3 (5.5 µg/mL). Acidified citrated plasma, citrated, CTAD or EDTA plasmas can be used. The assay is standardized with International standard 87/512. Assay time is only 60 minutes.

Kit contents include standards, high and low controls, coated plate, peroxidase-labeled antibody, buffers and ready to use TMB substrate.

Reagents, Storage and Stability

  • ELISA test strips: (12) with 8 wells each, precoated with tPA immobilized via a monoclonal anti tPA coating antibody; in an aluminium bag. Stable after opening until expiration date if stored 2-8°C with adhesive film in plastic bag with drying agent.
  • Washing buffer concentrate: (PBS; pH 7.3) containing detergent; 0.01% merthiolat; 1 bottle, 80 ml. Washing buffer (1+11.5 dilution of concentrate) is stable for 3 weeks if stored 2-8°C. Washing buffer concentrate is stable for 6 months after opening if stored 2-8°C after reconstitution.
  • Incubation buffer: (PBS; pH 7.3); contains stabilizer protein; 0.05% proclin; and dye, 1 bottle, 90 ml, ready for use. Stable after opening for 2 months if stored 2-8°C.
  • Calibrators: (Standards) numbered; lyophilized; 1 bottle each. Concentrations are lot dependent; consult label on the vial. Stable for 6 months if stored -20°C after reconstitution.
  • Control plasmas: “low level” and “high level” for checking purposes lyophilized; 1 bottle each. Concentrations are lot-dependent; consult the label on the vial. Stable for 6 months if stored -20°C after reconstitution.
  • Conjugate: monoclonal Anti-PAI-1-POX, dyed blue; 1 bottle, 0.3 ml. Stable after opening for 6 months if stored 2-8°C. A working solution is stable at room temperature for 60 minutes.
  • Chromogen TMB: (tetramethylbenzidine); 1 bottle, 12 ml; ready for use. Stable until expiration date after opening if stored 2-8°C.
  • Stopping solution: sulphuric acid 0.45 mol/l; 1 bottle 12ml; ready for use.
  • Adhesive film: for ELISA test strips (2).

The expiry date printed on the labels applies to storage of the unopened bottles at +2-8 °C.

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.