WO1993023561A1 - BIOIMMUNOESSAI POUR tPA ET PAI-1 - Google Patents

BIOIMMUNOESSAI POUR tPA ET PAI-1 Download PDF

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WO1993023561A1
WO1993023561A1 PCT/SE1993/000449 SE9300449W WO9323561A1 WO 1993023561 A1 WO1993023561 A1 WO 1993023561A1 SE 9300449 W SE9300449 W SE 9300449W WO 9323561 A1 WO9323561 A1 WO 9323561A1
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tpa
activity
sample
pai
solid phase
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PCT/SE1993/000449
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English (en)
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Steffen ROSÉN
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Chromogenix Ab
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins
    • G01N2333/8132—Plasminogen activator inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9726—Tissue plasminogen activator

Definitions

  • the present invention relates to a method of determin ⁇ ing the functional activity of the tissue plasminogen activator, tPA, which is a key enzyme in the fibrino- lytic system of the blood, and also to a method of estimating the activity of the physiologically most valuable inhibitor of this enzyme, designated PAI-1, where PAI stands for plasminogen activator inhibitor.
  • tPA tissue plasminogen activator
  • PAI-1 physiologically most valuable inhibitor of this enzyme
  • fibrinolytic system The purpose of the fibrinolytic system is to degrade fibrin clots, which is achieved by the enzyme plasmin.
  • Plasmin is produced in vivo, by activation of the proenzyme plasminogen. This activation will preferably take place locally, in that both plasminogen and plasminogen activators have a high affinity to fibrin clots, which, in turn, are formed in conjunction with trauma.
  • Solid fibrin can be replaced in vitro with other substances that have a high affinity to plasmin- ogen and plasminogen activators, for instance soluble fibrin (1) , cyanogen bromide degraded fibrinogen (2) or poly-D-lysine (3) , these substances functioning as stimulators in the tPA-dependent activation of plas ⁇ minogen.
  • the plasminogen can be activated by tPA and urokinase (u-PA) (see Chart 1) and possibly by some other enzyme that is associated with the so-called intrinsic pathway for the activation of plasminogen via FXII and callicrein. Chart 1.' An overview of the fibrinolysis system
  • tPA and also PAI-1 are released to the blood from endothelial cells.
  • both components have short lifetimes in plasma with half lives of less than 15 minutes.
  • the plasma concen ⁇ trations are low, in the case of tPA 2-20 ng/mL and in the case of PAI-15-50 ng/mL.
  • some individu ⁇ als constantly have clearly higher levels of PAI-1. It has been suggested that elevated levels of PAI-1 are predictors of arterial thrombosis in conj nction"with reinfarction (4) and of venous thrombosis in conjunc ⁇ tion with hip oint operations (5) .
  • PAI-1 is an effective inhibitor of tPA, illustrated by
  • One known method of achieving a good response is to measure tPA-activity indirectly via the activity of generated plasmin, measured either via degradation of fibrin - so-called euglobulin clot lysis time method (ECLT) or fibrin plate method (6) - or via the cleaving of chro ogenic (or fluorogenic) plasmin sub ⁇ strates containing p-nitroaniline (pNA) as chromophore (1, 2, 7).
  • ECLT euglobulin clot lysis time method
  • pNA p-nitroaniline
  • chromogenic plasmin substrates examples include H-D-Val-Leu-Lys-pNA (S-2251) , pyro-Glu-Phe-Lys-pNA (S2403) and Tos-Gly-Pro-Lys-pNA (Chromozyme PL) . See Chart 3."
  • the methods will be designed so that the recorded absorbance of released chromophore is related to the amount of tPA present in the sample.
  • bioimmunological method biological- assay, BIA
  • tPA is first extracted from a sample with the aid of surface-bound anti-tPA-antibod- ies.
  • BIA bioimmuno- assay
  • binding of tPA to antibodies does not eliminate the activity of all the bound enzyme.
  • plas ⁇ minogen and chromogenic plasmin substrate are then added in accordance with a known method, wherein the antibody-bound, enzymatically active tPA, activates plasminogen to plasmin, which thus cleaves the added plasmin substrate (8) .
  • S-2251 is used in this published method. Such activation does not normally require the presence of a substance that possesses fibrin-like properties with regard to its affinity to plasminogen and tPA for stimulating the reaction (8) .
  • One known method of eliminating the inhibition of tPA via PAI-1 in vitro involves lowering pH in conjunction with sampling to a pH value beneath 6, therewith interrupting the inhibition reaction (9, 10, 11).
  • suitable media in this regard are 1 mol/L acetate buffer, pH 3.9 (9) and 0.45 mol/L citrate, pH 4.4 (10).
  • essentially no denaturation of PAI- l occurs in this pH range.
  • a pH of about 3 (12) is required in this respect.
  • a typical procedure is to dilute acidified samples in a medium, normally water, which retains a low pH, and then mixing the sample with plasminogen and chromogenic plasmin substrate in the presence of a suitable stimulator according to the aforegoing and in a buffer which will provide a suit ⁇ able pH for plasminogen-activation and the activity of the plasmin formed during the activation process.
  • a suitable stimulator according to the aforegoing and in a buffer which will provide a suit ⁇ able pH for plasminogen-activation and the activity of the plasmin formed during the activation process.
  • pH ranges are 7-8.5.
  • the absorbency of released product p-nitroaniline from chromogenic plasmin substrate is then measured, said product constituting a measurement of the resultant plasmin activity, and therewith, indirectly, the tPA- activity in the sample.
  • the plasma sample used finally in the assay is normally diluted 100 times or more.
  • One method applied for eliminating interference of PAI-1 completely after neutralizing in conjunction with the addition of plasminogen and chromogenic plasmin substrate is additional acidification with HCI of the sample plasma obtained after centrifugation, therewith to obtain a pH of about 3, at which pH PAI-1 is denatured irreversibly according to the above (12) .
  • PAI-1 can be assayed specifically, either by immuno- logical methodology or by photometric methodology according to the aforegoing, preceded by an incubation stage in which a given quantity of tPA is incubated with a given sample volume, typically plasma, over a given period of time, whereafter the residual activity of tPA is determined.
  • the aforesaid ECLT method is used, to some extent, to estimate the global fibrinolytical activity. Sampling prior to this analysis is effected in a neutral tri- sodium citrate solution, which will thus not prevent tPA inhibition of PAI-1 in vitro. In brief, the method then proceeds as follows: The plasma sample is dilut ⁇ ed and weakly acidified with acetic acid, wherein a so-called euglobulin precipitate is formed. This precipitate contains, among other things, fibrinogen, tPA, plasminogen, prourokinase and PAI-1.
  • Time regis ⁇ tration commences from the moment that the fibrin clot or coagulate forms.
  • the clot acts as a stimulator to plasminogen activation and the plasmin formed will thus effect lysis of the fibrin clot.
  • a high concen ⁇ tration of active plasminogen activators will there ⁇ fore result in a short lysis time, and vice versa.
  • the ECLT method is best suited for the analysis of samples taken after venous stasis, which normally causes the release of tPA from endothelial cells and then pro ⁇ vides clot lysis times of about 10 min. and upwards. Greatly extended clot lysis times are a sign of low fibrinolytical activity. For practical reasons, the method is unsuitable for estimating the fibrinolytic activity in plasma samples that are taken prior to venous stasis, since the clot lysis times are- normally in the range of one to several hours.
  • Fibrinolytical activity in plasma samples taken prior to stasis is better estimated by the so-called fibrin plate method in which dissolved euglobulin precipitate is applied to a surface of a preformed fibrin clot, whereafter the diameter of the lysated zone is measured after incubating for many hours, normally over night. The diameter of the lysated zone forms a measurement of the fibrinolytic activity in the sample.
  • One feature common to both the ECLT method and the fibrin plate method is that they are not free from the influence of inhibitor activity and that they therefore give an estimation of the net activity of plasminogen activa- tors.
  • the methodologies are particularly difficult to standardize, because they include a number of measures of technical and biochemical com ⁇ plexity.
  • the published BIA tPA method has been found to correlate well with the ECLT method on plasma samples taken after venous stasis with blood collected in trisodium citrate (13) , and has been pro ⁇ posed as an alternative to the ECLT method with the important advantage of providing a method that can easily be standardized.
  • the method is highly sensitive and has a detection limit of about 10 mlU/mL, although it requires several hours incubation, normally overnight.
  • There has recently been described a method for the specific assaying of tPA-activity with the BIA tPA method in which sampling is effected in an acid environment, followed by additional acidification of plasma to denature PAI-1 (14) .
  • Binding of t-PA from the sample to the solid-phase bound anti-t-PA-antibody is effected in a neutral environment, similar to earlier published methods (8, 13) .
  • this recently reported BIA t-PA method (14) there is used a surface-bound monoclonal anti-tPA antibody which does not essentially influence the activity of tPA after binding to the antibody.
  • BIA tPA method (14) there is used a surface-bound monoclonal anti-tPA antibody which does not essentially influence the activity of tPA after binding to the antibody.
  • BIA tPA method there is no need to include a stimulator for the tPA-dependent plasminogen activation.
  • a common feature characteristic of all BIA t-PA methods published hitherto (8, 13, 14) is that binding of t-PA from the sample to the solid-phase bound anti t—PA-antibody is effected at a neutral pH. It is well known that a reduction in pH to about 6 will inhibit the reaction between t-PA and PAI-1 (9, 10). It is recommended for specific assaying of t-PA activity that the sample is acidified to pH 3 and therewith denature PAI-1 and thus prevent in vitro inhibition of t-PA, irrespective of whether the analysis is effected in an homogenous system (11, 12) or with the aid of the BIA t-P method (14).
  • PAI-l-activity can also be determined or estimated with respect to a sample in conjunction with tPA assays.
  • the present invention provides a method for determining the activity of tissue plasminogen activator (tPA) in a sample comprising a biological fluid, preferably a plasma sample, by (1) incubating the sample or a part thereof in the presence of a solid phase which binds tPA specifically, essentially without inhibiting its activity, tPA present in the sample being bound to the solid phase; and thereafter (2) determining the activity of tPA bound to the solid phase in a known manner; wherein, with the intention of essentially eliminating the influence of any plasminogen activitator inhibitor PAI-l, the incubation in step (1) is carried out at pH conditions which are controlled so as to inhibit the formation of a tPA/PAI-1 complex without denaturing PAI-l.
  • tPA tissue plasminogen activator
  • the tPA-activity of the sample is determined by dividing the sample into parts, whereafter
  • step (2) is carried out under equivalent conditions to determine the activity of the bound tPA for the first part (A) and for the second part (B) of the sample, the tPA-activity obtained for (B) divided by the tPA-activity obtained for (A) constituting a measurement of the PAI-l-activity in the sample.
  • the sample is comprised of whole blood or is comprised particularly of a plasma sample which is de ⁇ rived from a blood sample that has been collected under the same pH conditions as those used in step (1) of the method.
  • the pH value of the sample is adjusted in a step preceding step (1) to a value in which formation of a tPA/PAI-1 complex is inhibited.
  • the inventive method is thus a novel BIA-tPA method which, in comparison with earlier known methods of this kind, provides an essentially simpler method of assaying tPA-activity without being influenced by PAI-l, and also enables a simple estimation of the
  • PAI-l-activity to be made on the same analysis occa ⁇ sion, and therewith to discriminate between high and low levels of PAI-l.
  • a common feature of both proce ⁇ dures is that blood is collected in a medium which, after mixing, gives rise to a pH at which the reaction between tPA and PAI-l is inhibited but at which dena- turation of PAI-l is avoided.
  • the tPA-activity is then determined by bringing sample and the same or another medium into contact with a surface on which there is bound a substance that has an affinity to tPA, for instance an anti-tPA antibody, under conditions such as to obtain a pH that has the same characteristics as those described above with respect to mixing sampling medium and blood, the extraction of tPA in the permitted pH range being equivalent to extraction at a neutral pH.
  • a substance that has an affinity to tPA for instance an anti-tPA antibody
  • the tPA-activity is determined as disclosed in Chart 3, in the presence of or in the absence of tPA stimulator and with a plasmin substrate, which subsequent to reacting with plasmin releases detectable analyte, e.g. a chromogenic, fluorogenic or luminogenic plasmin substrate.
  • suitable pH values which favour plasminogen conversion and plasmin activity are pH 7.5-8.5.
  • the PAI-l-activity is estimated by bringing an acidified sample according to the aforegoing and another medium into contact with a surface on which there is bound a substance that has affinity to t ' PA, e.g. anti-tPA antibodies, under conditions such as to obtain a pH value of 6.3-9, suitably 7-8.5, which permits inhibition of tPA by complex-formation with PAI-l. Subsequent to incubation together with, e.g., the antibody-bound surface over an appropriate period of time, the tPA-activity is then determined in accor ⁇ dance with the description in the preceding paragrap .
  • a substance that has affinity to t ' PA e.g. anti-tPA antibodies
  • step (1) of the method are achieved by adding water, physiological saline or suitable buffer.
  • suitable buffer such as a TRIS buffer or a phosphate buffer.
  • the solid phase used to bind tPA in accordance with the invention is comprised conveniently of a solid carrier which is treated, preferably coated, with an appropriate substance, such as to impart to the carri- er the ability to bind tPA specifically without inhib ⁇ iting its activity.
  • a solid carrier which is treated, preferably coated, with an appropriate substance, such as to impart to the carri- er the ability to bind tPA specifically without inhib ⁇ iting its activity.
  • Such carriers are microtitre plates, beads, cylinders, latex particles, dispersions, etc. , of an appropriate solid material, for instance polypropylene or polystyrene.
  • Microtitre plates constitute a suitable carrier in accordance with the invention, and such plates are available commercially, for instance plates from Nunc, Roskilde, Denmark and Immulon from Dynatech.
  • the tPA binding substance is conveniently comprised of polyclonal or monoclonal antibodies that are bound to the surface of the carrier, for instance to the sur ⁇ faces of wells in a microtitre plate.
  • Preferably intact antibodies are used, particularly intact mono ⁇ clonal antibodies, although fragments thereof may also be used provided that the fragments exhibit desirable tPA binding ability.
  • the antibodies against tPA are of animal origin, suitably of mammal origin and may belong to different Ig classes. Monoclonal mouse or rabbit IgG antibodies against tPA may be used, for instance. Other examples of monoclonal anti-tPA antibodies are antibodies of human, bovine or murine origin.
  • Suitable monoclonal antibodies are available commer ⁇ cially, for instance the monoclonal anti-tPA antibody BIO from IMCO, Sweden.
  • the plasmin substrate used to determine tPA-activity in the sample may be comprised of any such substrate that is cleaved by plasmin in a manner that can be shown analytically.
  • the substrate will conveniently include a cleavable marker group which produces a discernible effect when cleaved.
  • Suitable substrates are chromogenic, fluorogenic or luminogenic substrat ⁇ es. Chromogenic substrates are particularly suitable, for instance the aforesaid chromogenic plasmin sub ⁇ strates.
  • Example l Tests illustrating equivalent binding of tPA at pH 6 and pH 8.
  • patient plasma was ana ⁇ lyzed in a Stabilyte medium having a tPA-activity of about 0.8 IU/mL and no significant PAI-l-activity.
  • the test was continued in precisely the same manner as in a) , although with a 30 min. incubation time of plasminogen and S-2403 instead of 20 min.
  • Example 2 Estimation of PAI-l-activity by measuring tPA-activity at the same time in plasma samples after binding tPA to anti-tPA antibodies at pH 6 and at pH 8.
  • Citrate samples were also used to assay PAI-l-activity with COATEST PAI (Chromogenix, M ⁇ lndal) while an assay of tPA-activity was made on plasma in Stabilyte medium with the BIA tPA method in microplates containing surface-bound monoclonal anti-tPA antibodies BIO, in accordance with the following:
  • tPA-activity was assayed simultaneously for each plasma sample at a pH of about 6 and at a pH of about 8, by mixing in a microplate well 100 ⁇ L water and 50 ⁇ L undiluted plasma sample (results in pH about 6) While 100 ⁇ L Tris-HCl, pH 8.4, 0.15 mol/L NaCI, 0.1% Tween 80 and 50 ⁇ L undiluted plasma sample (results in a pH of about 8) were mixed in another microplate well.
  • the Example shows a clear co-variation between the aforesaid ratio and PAI-activity, and also PAI-anti- gen. It is also seen that the ratio lies close to 1 at low PAI-l levels, which again indicates that binding of tPA at pH 6 is comparable with binding at pH 8.
  • Example 3 Analysis of tPA in whole blood at levels of relevance in thrombolytic therapy with rt-PA.
  • rt-PA Actilys, Boehringer Ingelheim
  • 5 ⁇ L rt-PA of a concentration of 50,000 IU/mL were added to 495 ⁇ L
  • 15 ⁇ L rt-PA of a concentration of 100,000 IU/mL were added to 485 ⁇ L.
  • 50 ⁇ L of the sample containing 500 IU/mL of rt-PA were then diluted with 200 ⁇ L of a physiological saline solution (0.9 percent by weight NaCI), while the sample containing 3,000 IU/mL was first diluted according to 50 ⁇ L sample + 450 ⁇ L physiological saline solution. 100 ⁇ L water and 50 ⁇ L of respective samples were then added to microplate wells containing surface-bound monoclonal antibodies B10 (IMCO, Sweden) . The tests were carried out in duplicate.
  • the invention can also be applied to whole blood and provides a quick method (the total analysis time being about 20 min.) of ob ⁇ taining a good estimation of the t-PA concentration in conjunction, for instance, with thrombolytic therapy.
  • Example 4 Comparison tests with different anti t-PA- antibodies for determining the t-PA-activity in BIA t- PA methodology in which t-PA is bound to a solid phase at pH 6.
  • Blood was collected from four test subjects in Stabi ⁇ lyte tubes in accordance with Example 3. The blood plasma obtained subsequent to centrifugation is then used to analyze the t-PA-activity in BIA t-PA systems with different solid-phase-bound anti t-PA-antibodies as catchers.
  • 100 ⁇ L 0.45 mol/L NaCI + 50 ⁇ L of respective plasma were added to each well of identical microplate wells containing surface-bound monoclonal anti t-PA ⁇ antibod-- ies 62E8 and 3B6 (Innogenetics, Antwerp, Belgium) .
  • 100 ⁇ L water + 50 ⁇ L t-PA standard (Actilyse, Boeh- ringer Ingelheim) having t-PA concentrations in the range of 0.05-1.25 IU/mL were added to similar wells.
  • the medium for t-PA standard solutions was 0.1 mol/L Tris-HCl, pH 8.4, 0.1% Tween 80. Subsequent to binding for 60 min.
  • the wells were rinsed with 3 x 300 ⁇ L 0.1 mol/L Tris-acetate, pH 8.4, 0.1% Tween 80, whereafter there were added 150 ⁇ L of a mixture con ⁇ taining plasminogen, 0.15 mg/mL, and chromogenic plasmin substrate S-2403, 1 mmol/L in a medium con ⁇ taining 0.1 mol/L Tris-acetate, pH 8.3, 0.1% Tween 80. Subsequent to incubation for 55 min. at 37°C, the absorbance was recorded at 405 nm with a reference wavelength of 490 nm and the contents of the samples were read-off against a standard curve.
  • the valid tPA- activity methods earlier published have either employed much stronger dilution of the sample (1, 7, 12) or, in the case of the BIA tPA method with weak dilution of the sample, additional acidification of plasma is employed to denature PAI-l, whereafter the sample is neutralized so that the immuno-adsorption takes place at a neutral pH to a slightly alkaline pH (14) .
  • the invention affords valid assaying of the tPA- activity with a simplicity not earlier achieved and affords a high degree of sensitivity enabling detection of a tPA-activity of 10 mlU/mL subsequent to incubation for 60 min. at 37°C.

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Abstract

Procédé in vitro servant à analyser l'activité de tPA, combiné éventuellement à l'analyse de l'activité de PAI-1. Le procédé est caractérisé par le fait qu'il comprend les étapes (i) d'incubation d'un échantillon contenant tPA avec une phase solide liant spécifiquement tPA dans des conditions de pH inhibant la formation d'un complexe tPA-PAI-1, ainsi que (ii) de détermination de l'activité de tPA de la phase solide. Les conditions de pH signifient que PAI-1 ne sera pas dénaturé de façon irréversible. Si PAI-1 doit être déterminé fonctionnellement, l'échantillon est conditionné par rapport au pH avant l'étape (i). Une première partie et une deuxième partie de l'échantillon sont ensuite soumises à incubation en fonction de l'étape (i) à l'exception des conditions de pH pour la deuxième partie, qui sont sélectionnées de façon à former un complexe PAI-1-tPA. L'activité de tPA lié à la phase solide est déterminée séparément pour les deux parties en tant qu'étape finale et l'activité de PAI-1 est calculée à partir du rapport entre les mesures de l'activité de tPA des différentes parties de l'échantillon.
PCT/SE1993/000449 1992-05-21 1993-05-19 BIOIMMUNOESSAI POUR tPA ET PAI-1 WO1993023561A1 (fr)

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SE9201615A SE9201615L (sv) 1992-05-21 1992-05-21 Bioimmunologisk metod för bestämning av tPa och PAI-1
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001452A1 (fr) * 1993-06-28 1995-01-12 Akzo Nobel N.V. Quantification de l'inhibiteur de l'activateur du plasminogene de type 1 actif

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0298783A1 (fr) * 1987-07-10 1989-01-11 Yamanouchi Pharmaceutical Co. Ltd. Méthode, dispositif et kit de test pour la détermination de l'activité d'un activateur tissulaire du plasminogène
WO1989000005A1 (fr) * 1987-07-06 1989-01-12 Cytrx Biopool Ltd. Analyses ameliorees d'activateur plasminogene et d'inhibiteur d'activateur plasminogene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000005A1 (fr) * 1987-07-06 1989-01-12 Cytrx Biopool Ltd. Analyses ameliorees d'activateur plasminogene et d'inhibiteur d'activateur plasminogene
EP0298783A1 (fr) * 1987-07-10 1989-01-11 Yamanouchi Pharmaceutical Co. Ltd. Méthode, dispositif et kit de test pour la détermination de l'activité d'un activateur tissulaire du plasminogène

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 72, EMBASE, Dialog Accession No. 8576490, EMBASE Accession No. 92252531, MEIJER P. et al., "Bioimmunoassay for Tissue-Type Plasminogen Activator (t-PA) in Human Plasma: Evaluation of Blood Sampling and Handling Procedures and Comparison with Other t-PA Activity Methods"; & FIBRINOLYSIS (UNITED KINGDOM), 1992, 6/Suppl. 3(97-99). *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001452A1 (fr) * 1993-06-28 1995-01-12 Akzo Nobel N.V. Quantification de l'inhibiteur de l'activateur du plasminogene de type 1 actif
US5753457A (en) * 1993-06-28 1998-05-19 Akzo Nobel N.V. Quantification of active plasminogen-activator-inhibitor-type-1

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