The Technozym® tPA Combi Actibind® ELISA test kit measures t-PA functional activity, non-complexed t-PA antigen and t-PA-PAI-1 complexes. It is not affected by the presence of other plasminogen activators. The measurement range is 0.05-10 IU/mL for t-PA activity and 0.1-20 ng/mL for t-PA antigen. The inter- and intra-assay variations are less than 10 % and 5 %, respectively.

Determination: 42 samples in duplicate

  1. PLATE + PLATE COVER 12 x 8 well plastic microtitre strips precoated with a monoclonal anti-t-PA coating antibody in bicarbonate buffer, 1% bovine serum albumin (BSA), (TC-Code GN).
  2. STANDARD 1 x lyophilized recombinant t-PA, calibrated against NIBSC 86/670 (TC-Code AW).
  3. POX-ANTIBODY 1 x conjugated anti t-PA antibody (concentrated), 0,3ml . (TC-Code KM).
  4. INCUBATION BUFFER 1×90 ml (PBS; pH 7,3) contains stabiliser protein, 0,05% proclin and blue dye, 1bottle, , ready for use (TC-Code NB)
  5. SUBSTRATE – (green cap) 1x 12 mL TMB (tetramethylbenzidine) (TC-Code KN) ready to use.
  6. STOP SOLUTION – (red cap) 1x 15 mL sulphuric acid 0.45 mol/l (TC-Code KK) ready to use.
  7. WASHING BUFFER – concentrate (1+11,5) 1x80ml (PBS pH 7,3) containing detergent, 0,01 % merthiolat (TC- Code NA).
  8. PLASMINOGEN ACTIVATOR DETECTION MIXTURE. 1 x lyophilized H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt, Gluplasminogen and Cyanogen bromide fibrinogen fragments (TC Code BA).
  9. PLASMIN-ACTIVATOR DETECTION MIXTURE DILUTION BUFFER (white cap) 1 x 20 mL 50mM Tris, 12mM NaCl. Ready to use (TC-Code DA).

Also required

  1. Micropipettes and a multichannel micropipette; pipette tips.
  2. Glass or plastic test tubes for diluting the standard + samples.
  3. Laboratory bottles or beakers and graduated cylinders for diluting wash and incubation buffer.
  4. Distilled or deionised water.
  5. Absorbent paper towels.
  6. Microtitre plate washer (alternatively, washing can be performed manually using a multichannel pipette or repeating syringe).
  7. A microtitre reader equipped with a 405 nm filter and, if possible, a 492 nm reference filter.
  8. A 37° C incubator
  9. Graph paper.

In the Actibind t-PA test, an antibody, which does not interfere with t-PA functional activity, is coated onto a microtitre plate and used to bind t-PA contained in plasma to the plate surface.

Following an incubation period, non-bound plasma components are washed away and an activity substrate solution containing Glu-plasminogen, CNBr-fragments of fibrinogen and a chromogenic plasmin substrate is incubated in the plate. The bound t-PA activates Glu-plasminogen to yield plasmin. The reaction between plasmin and the chromogenic plasmin substrate releases a coloured product whose concentration is proportional to the amount of active t-PA in the test sample.

After photometrically measuring this reaction, the microtitre plate is washed to remove the activity substrate solution. The t-PA antigen remains bound to the plate. A horseradish peroxidase (POX) conjugated monoclonal anti-t-PA antibody which recognizes both active, t-PA and inactive t-PA is then incubated on the plate. Following incubation and washing of the plate, a POX substrate is used to produce a coloured reaction product whose concentration is proportional to the total t-PA content of the test sample.

Tissue plasminogen activator (t-PA) is a 70,000 dalton glycoprotein which serves as the major activator of the blood fibrinolytic system. It is synthesized principally by endothelial cells and released into the blood stream following stimulus, e.g. exercise or venous occlusion. t-PA is unique among the plasminogen activators due to the fact that its action is highly fibrin specific. Only in the presence of fibrin, to which it binds, does t-PA efficiently activate Glu-plasminogen to yield the cleavage product, plasmin. Plasmin, in turn, degrades fibrin, the insoluble protein polymer in thrombi.

tPA circulates in plasma at a concentration of approximately 2-8 ng/mL. However, 95 % of circulating t-PA is complexed with the plasminogen activator inhibitor, PAI-1, and thus in an inactive form. Upon venous occlusion the concentration of t-PA antigen increases to 15 ng/mL or higher and t-PA’s activity rises accordingly. This increase is probably due to a combination of increased t-PA release by endothelial cells and a reduction in t-PA clearance from the occlusion site.t-PA activity can only be measured in samples from which inhibitors of t-PA have been removed.In the Actibind system this is simply accomplished by specific immunoabsorption of the tPA contained in the plasma sample by an antibody immobilized onto a microtitre plate surface. In this first step t-PA and t-PA-PAI-1-complexes are bound to the plate and active, non-complexed t-PA is therafter quantified using a solution of Glu-plasminogen, fibrinogen fragments and a synthetic substrate which reacts with plasmin formed in the reaction to yield a coloured reaction product. Low activity in this first step can be due to either a decrease in the quantity of total t-PA bound to the plate or to an excess of PAI-1 in the plasma sample, which inactivates t-PA and leads to the formation of t-PA-PAI-1-complexes. To differentiate between these two reasons for decreased t-PA function, the second part of the Actibind assay is then used to determine the total amount of t-PA antigen bound onto the microtiter plate through the use of a peroxidase-labelled monoclonal antibody.

 

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
Component Molecular
Weight kDa
Plasma
Concentration
mg/l
Plasma
Concentration
μmol/l
Fibrinogen 330 3000 9
Prothrombin 72 150 2
Factor V 330 20 0.05
Factor VII 50 0.5 0.01
Factor VIII 330 0.1 0.0003
Factor IX 56 5 0.09
Factor X 59 8 0.13
Factor XI 160 5 0.03
Factor XII 80 30 0.4
Factor XIII 320 10 0.03
Protein C 62 4 0.06
Protein S 70 10 (free) 0.14
Protein Z 62 2 0.03
Prekallikrein 86 50 0.6
HMW kininogen 120 70 0.6
Fibronectin 450 300 0.7
Plasminogen 92 200 2
t-PA 60 0.005 0.0001
Urokinase 53 0.004 0.0001
Antithrombin 58 145 2.5
Heparin Cofactor II 66 80 1.2
Plasmin Inhibitor 63 60 1
Protein C Inhibitor 57 4 0.07
α2-Macroglobulin 725 2000 3