Technozym® t-PA Antigen EDTA ELISA is a highly sensitive complete sandwich ELISA kit for the quantitative determination of Tissue Plasminogen Activator (tPA) in human plasma. The assay is a double sandwich ELISA employing two monoclonal antibodies. The system detects both complexed and uncomplexed tPA. Detection limit is 1 ng/mL. Acidified citrated plasma, citrated, CTAD or EDTA plasmas can be used. The assay is standardized with the International standard 98/714. Assay time is 2.5 hours. Kit contents include standards, high and low controls, coated plate, peroxidase-labeled antibody, buffers and ready to use TMB substrate. The sample buffer contains EDTA for increased recovery of t-PA in in normal samples.
- ELISA test strips (12) with 8 wells each, coated anti t-PA monoclonal antibody; the drying agent is supplied in an aluminium bag.
- Washing buffer concentrate (PBS; pH 7.3); containing detergent; 0.01% merthiolate; 1 bottle, 80 ml.
- Incubation buffer (PBS; pH 7.3); contains stabiliser protein; 0.05% proclin; and blue dye, 1 bottle, 90 ml, ready for use.
- Sample dilution buffer (PBS, EDTA); contains stabiliser protein; 1 bottle, 20mL
- Calibrators (Standards) numbered; lyophilised; 1 bottle each. Concentrations are lot-dependent; consult label on the vial.
- Control plasmas “low level” and “high level” for checking purposes lyophilised; 1 bottle each. Concentrations are lot-dependent; consult the label on the vial.
- Conjugate monoclonal Anti-t-PA POX; dyed blue; 1 bottle, 0.3 ml.
- Chromogen TMB (tetramethylbenzidine); 1 bottle, 12 ml; ready for use.
- Stopping solution sulphuric acid 0.45 mol/l; 1 bottle 12ml; ready for use.
- Adhesive film: for ELISA test strips (2).
MATERIAL REQUIRED (but not supplied with the kit)
- Distilled water
- Test tubes for diluting standard and samples
- Measuring cylinder (100mL and 1000 ml)
- Precision pipettes (10, 100 and 1000 μl)
- Variable pipette (1000 μl)
- Multichannel and/or dispensing pipettes (100 and 200 μl)
- ELISA washer or multichannel pipette
- ELISA reader with 450 nm filter, with a 620 nm reference filter if available.
- Incubator (37 °C)
Normal range for t-PA antigen is between 2- 8 ng/ml It is recommended that individual laboratories establish their own ranges. For plasma obtained after venous occlusion values greater than +10ng/ml, should be obtained.
A failure of t-PA levels to increase upon venous occlusion is indicative of an impaired fibrinolytic capacity and may indicate thrombotic tendencies.
The calibration material used is the WHO international standard for tissue plasminogen activator (t-PA).
|What is the difference between single and two-chain tPA?|
|Single-chain tPA is the native form of tPA secreted from endothelial cells, whereas the two-chain form is the result of the proteolytic activity of plasmin. Both forms are catalytically active and have similar enzymatic properties in the presence of fibrin.|
|Explain the function of t-PA.|
|tPA activates plasminogen to plasmin, and also binds to fibrin. It is synthesized mainly in vascular endothelial cell and is secreted into the plasma continuously, and also through the acute release of tPA. tPA is a relatively poor activator of plasminogen in the absence of fibrin due to the low affinity of tPA for its substrate. However, tPA has a high affinity for fibrin, and binding increases its activating capacity up to 1000 fold. This dramatic increase is attributed to specific binding sites on the fibrin surface that concentrate and correctly orientate tPA with its substrate, as well as promote efficient clot lysis.|
|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.|
|Which substrate is best suited for measuring two-chain tPA, and why?|
|S-2765™, S-2366™, and S-2288™, S-2403™ are suitable for single-chain tPA, and S-2251™ was used in the discontinued Chromogenix Coaset® tPA kit, but there are no substrates specifically for two-chain tPA. A paper by Verheijen et al. (Thromb Res, 1985; 39: 281 – 288), however, describes a method comparing the direct amidolytic activity of tPA on S-2366™ and the plasminogen activating activity. Also, the substrate S-2288™ is suitable for measuring double-chain tPA because it has a slightly higher sensitivity than S-2366™. S-2288™ should be used with purified systems, though, since this substrate is sensitive for several proteases.|
|Protein concentrations in plasma|