EP1155328A1 - Test kit for analysing factor viii-splitting protease - Google Patents

Test kit for analysing factor viii-splitting protease

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Publication number
EP1155328A1
EP1155328A1 EP00908814A EP00908814A EP1155328A1 EP 1155328 A1 EP1155328 A1 EP 1155328A1 EP 00908814 A EP00908814 A EP 00908814A EP 00908814 A EP00908814 A EP 00908814A EP 1155328 A1 EP1155328 A1 EP 1155328A1
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EP
European Patent Office
Prior art keywords
willebrand factor
von willebrand
test kit
collagen
splitting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00908814A
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German (de)
French (fr)
Inventor
Helena E. Gerritsen
Miha Furlan
Peter Turecek
Katalin Varadi
Jürgen SIEKMANN
Bernhard LÄMMLE
Hans-Peter Schwarz
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Baxter AG
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Baxter AG
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Publication date
Application filed by Baxter AG filed Critical Baxter AG
Publication of EP1155328A1 publication Critical patent/EP1155328A1/en
Withdrawn legal-status Critical Current

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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]

Definitions

  • the present invention relates to a test kit and a method for determining the protease splitting von Willebrand factor and the application of the method for the differential diagnosis of thrombotic thrombocytopenic purpura (TTP) and the hemolytic-uremic syndrome (HUS).
  • TTP thrombotic thrombocytopenic purpura
  • HUS hemolytic-uremic syndrome
  • a test kit consisting of a von Willebrand factor standard preparation, which is free of Willebrand factor-splitting activity, as a substrate for the von Willebrand factor-splitting activity in a sample or in the patient plasma and a system for quantitative determination of the binding of von Willebrand factor to collagen enables the analysis of the von Willebrand factor-splitting protease and the differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic-uraemic syndrome for the first time in a simple manner.
  • the von Willebrand factor standard preparation can be inactivated specifically with regard to Willebrand factor-splitting protease, but a generally protease-inactivated von Willebrand factor standard preparation can also be used.
  • the quantitative determination of the collagen binding activity is carried out on immobilized avid collagen, which is preferably present bound to a microtiter plate. It is advantageous if the immobilized .collagen is covalently bound.
  • the avide collagen is an enzymatically degraded, soluble, human collagen.
  • the von Willebrand factor standard preparation is preferably a human reference plasma, in which the protease activity cleaving by Willebrand factor is inactivated.
  • the von Willebrand factor standard preparation is a recombinant von Willebrand factor.
  • the present invention also relates to a method for the detection of an acquired or congenital deficiency of the protease splitting von Willebrand factor, wherein a preparation prepared by Willebrand factor factor, which is free of Willebrand factor splitting activity, as a substrate with that of Willebrand factor -cleaving activity of the patient's plasma or dilutions of the same is brought into contact and after incubation the quantitative detection of the residual von Willebrand factor is carried out by its binding properties to collagen.
  • dilutions of patient and normal plasmas were activated with barium and mixed with undiluted vWF substrate.
  • a protease-inactivated plasma was used as the substrate.
  • the reaction was stopped and the incubation mixture was placed on an ELISA plate which had previously been coated with collagen.
  • the long vWF multimers bind very strongly to the collagen, the small molecules hardly or not at all.
  • the supernatant is then discarded and the vWF molecules which have been bound to the collagen can be detected by means of generally known methods from the prior art, for example stained with the aid of peroxidase-labeled antibodies.
  • FIG. 6 shows the determination of the vWF-cleaving protease in a patient with thrombotic thrombocytopenic purpura during the therapeutic treatment
  • the different affinity of the different long vWF multimers is used in the test according to the invention (FIG. 1).
  • the long multimers have a very high affinity for collagen, while the small molecules have little or no affinity.
  • the vWF consists of different long multimers from the same subunit.
  • the subunits are connected to each other at the carboxy and at the amino termini by disulfide bridges (FIG. 2).
  • disulfide bridges FIG. 2
  • each subunit can be cleaved by the vWF protease (FIG. 3).
  • FIG. 5 shows the studies on two families whose members suffer from hereditary TTP.
  • the immunoblotting method is shown above, and the values obtained using the collagen binding assay according to the invention (CBA) are shown below.
  • CBA collagen binding assay according to the invention
  • the advantage of the method according to the invention is the possibility of quantitative evaluation and the simpler and, above all, shorter feasibility, which is of crucial importance in the acute diagnosis of the life-threatening clinical pictures.
  • a particularly advantageous embodiment of the test according to the invention uses the collagen binding assay according to AT 403 853 for the quantitative determination of the protease substrate by von Willebrand factor.
  • Another application of the present invention consists in the quantitative determination of the protease splitting von Willebrand factor during purification and characterization as a therapeutic plasma fraction or for quality control of therapeutically used full plasmas. This is explained in more detail by the following example (see Fig. 7):
  • the protease (multimerase) cleaving factor by Willebrand was partially purified according to AT 404 359.
  • the Multimerasepr was paration on a column packed with Fractogel ® TSK AF-Orange (Merck), and eluted with a buffer gradient of pH 5.5 - pH 8.5 in Tris buffer (10 mM Tris, 10 mM Na 3rd citrate, 150 mM NaCl) eluted.
  • the UV absorption at 280 nm (total protein) and the collagen binding activity of a von Willebrand factor standard were measured after incubation with the fractions according to the invention.
  • the reciprocal of the collagen binding activity was entered in the elution diagram and shows that in addition to a part of the protease activity which is in the column run (fraction 10-20), the majority of the activity could be e

Abstract

The invention relates to a test kit for analysing the von Willebrand factor-splitting protease and for carrying out differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with haemolytic-uraemic syndrome. Said test kit consists of a standard von Willebrand factor preparation which is free of von Willebrand factor-splitting activity, as a substrate for the von Willebrand factor-splitting activity in a sample or in the patient plasma, and a system for quantitatively determining the bonding of von Willebrand factor to collagen. The invention also relates to a method for detecting an acquired or congenital deficiency of von Willebrand factor-splitting protease.

Description

TESTKIT ZUR ANALYTIK DER FAKTOR-VIII-SPALTENDEN PROTEASE ANALYTICAL TEST KIT FOR THE FACTOR VIII-SPLITTING PROTEASE
Die vorliegende Erfindung betrifft einen Testkit und ein Verfahren zur Bestimmung der von Willebrand Faktor-spaltenden Protease sowie die Anwendung des Verfahrens zur Differentialdiagnostik der thrombotisch thrombozytopenischen Purpura (TTP) und des hä- molytisch-urämischen Syndroms (HUS) .The present invention relates to a test kit and a method for determining the protease splitting von Willebrand factor and the application of the method for the differential diagnosis of thrombotic thrombocytopenic purpura (TTP) and the hemolytic-uremic syndrome (HUS).
Mangel an von Willebrand Faktor-spaltender Protease wurde bei Patienten mit TTP festgestellt, während Patienten mit hämolytisch-urämischem Syndrom (HUS) eine normale Aktivität der von Willebrand Faktor-spaltenden Protease aufwiesen. Abgesehen von den bisher bereits beschriebenen verschiedenen aufwendigen Verfahren (Blood 1996; 87:4223) ist zur Zeit kein Bestimmungsverfahren der Aktivität der von Willebrand Faktor-spaltenden Protease verfügbar. Ein einfaches Bestimmungsverfahren würde die Unterscheidung zwischen TTP und HUS erleichtern und so ein besseres Monitoring der Therapie von Patienten mit TTP erlauben.Deficiency of von Willebrand factor-splitting protease was found in patients with TTP, while patients with hemolytic-uremic syndrome (HUS) showed normal activity of the von Willebrand factor-splitting protease. Apart from the various complex methods already described (Blood 1996; 87: 4223), there is currently no method available for determining the activity of the protease that cleaves von Willebrand factor. A simple determination procedure would make it easier to distinguish between TTP and HUS and thus allow better monitoring of the therapy of patients with TTP.
Es wurde nun überraschenderweise gefunden, dass ein Testkit, bestehend aus einer von Willebrand Faktor-Standardpräparation, die frei von von Willebrand Faktor-spaltender Aktivität ist, als Substrat für die von Willebrand Faktor-spaltende Aktivität in einer Probe oder im Patientenplasma und einem System zur quantitativen Bestimmung der Bindung von von Willebrand Faktor an Collagen die Analytik der von Willebrand Faktor-spaltenden Protease und die Differentialdiagnostik zwischen Patienten mit thrombotisch thrombozytopenischer Purpura und Patienten mit hämolytisch-urämischem Syndrom erstmals auf einfache Weise ermöglicht. Die von Willebrand Faktor-Standardpräparation kann dabei sowohl spezifisch hinsichtlich von Willebrand Faktor-spaltender Protease inaktiviert sein, es kann dabei aber ebenso eine generell Protease-inaktivierte von Willebrand Faktor-Standardpräparation verwendet werden.It has now surprisingly been found that a test kit consisting of a von Willebrand factor standard preparation, which is free of Willebrand factor-splitting activity, as a substrate for the von Willebrand factor-splitting activity in a sample or in the patient plasma and a system for quantitative determination of the binding of von Willebrand factor to collagen enables the analysis of the von Willebrand factor-splitting protease and the differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic-uraemic syndrome for the first time in a simple manner. The von Willebrand factor standard preparation can be inactivated specifically with regard to Willebrand factor-splitting protease, but a generally protease-inactivated von Willebrand factor standard preparation can also be used.
Gemäß einer bevorzugten Ausführungsform des erfindungsgemäßen Testkits erfolgt die quantitative Bestimmung der Collagen-Bindungsaktivität an immobilisiertem aviden Collagen, welches vorzugsweise an eine Mikrotiterplatte gebunden vorliegt. Günstig ist dabei, wenn das immobilisierte .Collagen kovalent gebunden ist .According to a preferred embodiment of the test kit according to the invention, the quantitative determination of the collagen binding activity is carried out on immobilized avid collagen, which is preferably present bound to a microtiter plate. It is advantageous if the immobilized .collagen is covalently bound.
Weiters ist dabei vorteilhaft, wenn das avide Collagen ein enzy- matisch abgebautes, lösliches, humanes Collagen ist.It is also advantageous if the avide collagen is an enzymatically degraded, soluble, human collagen.
Vorzugsweise ist die von Willebrand Faktor-Standardpräparation ein humanes Referenzplasma, bei welchem die von Willebrand Faktor-spaltende Proteaseaktivität inaktiviert ist.The von Willebrand factor standard preparation is preferably a human reference plasma, in which the protease activity cleaving by Willebrand factor is inactivated.
Gemäß noch einer weiteren Ausführungsform ist vorgesehen, dass die von Willebrand Faktor-Standardpräparation ein reko binanter von Willebrand Faktor ist.According to yet another embodiment, it is provided that the von Willebrand factor standard preparation is a recombinant von Willebrand factor.
Günstig ist auch, wenn die von Willebrand Faktor-Standardpräparation ein von Willebrand-Faktor-Dimer ist.It is also favorable if the von Willebrand factor standard preparation is a von Willebrand factor dimer.
Weiters betrifft die vorliegende Erfindung auch noch ein Verfahren zum Nachweis eines erworbenen oder kongenitalen Mangels der von Willebrand Faktor-spaltenden Protease, wobei eine von Willebrand Faktor-Standardpräparation, die frei von von Willebrand Faktor-spaltender Aktivität ist, als Substrat mit der von Willebrand Faktor-spaltenden Aktivität des Patientenplasmas oder Verdünnungen desselben in Kontakt gebracht wird und nach Inkubation die quantitative Detektion des residualen von Willebrand Faktors durch seine Bindungseigenschaften an Collagen erfolgt.Furthermore, the present invention also relates to a method for the detection of an acquired or congenital deficiency of the protease splitting von Willebrand factor, wherein a preparation prepared by Willebrand factor factor, which is free of Willebrand factor splitting activity, as a substrate with that of Willebrand factor -cleaving activity of the patient's plasma or dilutions of the same is brought into contact and after incubation the quantitative detection of the residual von Willebrand factor is carried out by its binding properties to collagen.
Die beiliegenden Abbildungen zeigen das erfindungsgemäße Test- prinzip und die Anwendung zur Diagnostik von Patientenplasmen. Die Übereinstimmung der komplizierten SDS-Agarose gelelektropho- retischen Qualifizierung mit anschließendem Immunblotting mit der quantitativen Methode des erfindungsgemäßen Collagen-Bin- dungstestε (CBA) ist ebenfalls in den beiliegenden Abbildungen dargestellt .The accompanying figures show the test principle according to the invention and the application for the diagnosis of patient plasmas. The agreement of the complicated SDS agarose gel electrophoresis qualification with subsequent immunoblotting with the quantitative method of the collagen binding test (CBA) according to the invention is also shown in the accompanying figures.
Erfindungsgemäß wurden Verdünnungen von Patienten- und Normal- plasmen mit Barium aktiviert und mit unverdünntem vWF-Substrat versetzt. Als Substrat wurde ein Protease-inaktiviertes Plasma benutzt. Nach zwei Stunden Verdauung in Anwesenheit von Urea wurde die Reaktion gestoppt und das Inkubationsgemisch auf eine ELISA-Platte gegeben, die zuvor mit Collagen beschichtet worden war. Die langen vWF-Multimere binden sehr stark an das Collagen, die kleinen Moleküle kaum oder gar nicht . Der Oberstand wird sodann verworfen und die vWF-Moleküle, die an das Collagen gebunden wurden, können mittels allgemein bekannter Verfahren des Standes der Technik detektiert werden, z.B. mit Hilfe Peroxida- se-markierter Antikörper angefärbt werden.According to the invention, dilutions of patient and normal plasmas were activated with barium and mixed with undiluted vWF substrate. A protease-inactivated plasma was used as the substrate. After two hours of digestion in the presence of urea the reaction was stopped and the incubation mixture was placed on an ELISA plate which had previously been coated with collagen. The long vWF multimers bind very strongly to the collagen, the small molecules hardly or not at all. The supernatant is then discarded and the vWF molecules which have been bound to the collagen can be detected by means of generally known methods from the prior art, for example stained with the aid of peroxidase-labeled antibodies.
Die vorliegende Erfindung wird nun unter Bezugnahme auf die beiliegenden Abbildungen näher erläutert .The present invention will now be explained in more detail with reference to the accompanying figures.
Es zeigen:Show it:
Fig. 1 das Prinzip des erfindungsgemäßen Kollagen-Bindungstests,1 shows the principle of the collagen binding test according to the invention,
Fig. 2 die Wirkung der Protease mit vWF-spaltender Aktivität,2 shows the effect of the protease with vWF-cleaving activity,
Fig. 3 die Wirkung der Protease mit vWF-spaltender Aktivität auf die vWF-Untereinheit,3 shows the effect of the protease with vWF-cleaving activity on the vWF subunit,
Fig. 4 die Testkalibrierung,4 shows the test calibration,
Fig. 5 die Bestimmung der vWF-spaltenden Protease in Patienten mit thrombotisch thrombozytopenischer Purpura,5 the determination of the vWF-cleaving protease in patients with thrombotic thrombocytopenic purpura,
Fig. 6 die Bestimmung der vWF-spaltenden Protease in einem Patienten mit thrombotisch thrombozytopenischer Purpura während der therapeutischen Behandlung, und6 shows the determination of the vWF-cleaving protease in a patient with thrombotic thrombocytopenic purpura during the therapeutic treatment, and
Fig. 7 die quantitative Bestimmung der vWF-spaltenden Protease in gereinigten Plasmafraktionen.7 shows the quantitative determination of the vWF-cleaving protease in purified plasma fractions.
In dem erfindungsgemäßen Test (Fig. 1) wird die unterschiedliche Affinität der verschiedenen langen vWF-Multimere ausgenützt. Die langen Multimere haben eine sehr hohe Affinität zu Collagen, während die kleinen Moleküle keine oder nur geringe Affinität aufweisen. Der vWF besteht aus verschiedenen langen Multimeren derselben Untereinheit. Die Untereinheiten sind jeweils an den Carboxy- und an den Aminotermini durch Disulfidbrücken miteinander verbunden (Fig. 2) . Jede Untereinheit kann bei Tyrosin842-Methio- nin843 durch die vWF-Protease gespalten werden (Fig. 3) .The different affinity of the different long vWF multimers is used in the test according to the invention (FIG. 1). The long multimers have a very high affinity for collagen, while the small molecules have little or no affinity. The vWF consists of different long multimers from the same subunit. The subunits are connected to each other at the carboxy and at the amino termini by disulfide bridges (FIG. 2). In tyrosine842-methionine843, each subunit can be cleaved by the vWF protease (FIG. 3).
Unter diesen Bedingungen wurde mit verschiedenen Normalplasmaverdünnungen eine Eichkurve erstellt (Fig. 4) . Auf der Y-Achse ist die Extinktion bei 492 nm aufgetragen, auf der X-Achse die Normalplasmaverdünnungen. Es wird angenommen, dass eine Verdünnung von Normalplasma von 1/20 einer Aktivität von 100% entspricht. Je mehr vWF-spaltende Protease im Gemisch vorhanden ist, desto stärker wird der vWF verdaut, desto weniger kann er an Collagen binden und um so kleiner wird die Extinktion.Under these conditions, a calibration curve was created using various normal plasma dilutions (FIG. 4). The absorbance at 492 nm is plotted on the Y axis and the normal plasma dilutions on the X axis. It is believed that 1/20 dilution of normal plasma corresponds to 100% activity. The more vWF-cleaving protease is present in the mixture, the more the vWF is digested, the less it can bind to collagen and the lower the extinction.
In Fig. 5 sind die Untersuchungen an zwei Familien dargestellt, deren Mitglieder an hereditärer TTP leiden. Oben ist die Immun- blotting-Methode, unten die Werte, die mit dem erfindungsgemäßen Collagen-Bindungsassay (CBA) erhalten wurden, dargestellt. Ganz links ist der Propositus, der homozygot defizient an vWF-spaltender Protease ist. Als nächster kommt der Bruder, ebenfalls homozygot defizient, die Schwester normal, beide Eltern obligatorisch heterozygot . Dort, wo man in der Immunblotting-Methode einen stark verdauten vWF sieht, erhielt man auch im erfindungsgemäßen CBA einen Wert um 100%, dort wo das Substrat nicht verdaut wurde, konnte auch im erfindungsgemäßen CBA keine Aktivität festgestellt werden. Rechts sind Mitglieder derselben Familie, die alle homozygot defizient sind.FIG. 5 shows the studies on two families whose members suffer from hereditary TTP. The immunoblotting method is shown above, and the values obtained using the collagen binding assay according to the invention (CBA) are shown below. On the far left is the propositus, which is homozygous for vWF-cleaving protease. Next comes the brother, also homozygous deficient, the sister normal, both parents obligatory heterozygous. Wherever a strongly digested vWF is seen in the immunoblotting method, a value of around 100% was also obtained in the CBA according to the invention, and no activity was found in the CBA according to the invention where the substrate was not digested. On the right are members of the same family, all of whom are homozygously deficient.
Außerdem wurden auch Plasmen eines Patienten mit hereditärer TTP während der Therapie mit FFP untersucht (Fig. 6) . Oben ist wiederum die Immunblotting-Methode, unten sind die Werte, die mit dem erfindungsgemäßen CBA erhalten wurden, zu sehen. Ganz links ist eine Plasmaprobe aufgeführt, die vor der Therapie entnommen wurde. Die weiteren Proben sind nach einem ersten und einem zweiten Plasmaaustausch entnommen worden. Sowohl bei der Immunblotting-Methode als auch bei dem erfindungsgemäßen Collagen- Bindungsassay kann eine Zunahme der Aktivität nach dem ersten Austausch, ein weiterer Anstieg nach der zweiten und danach eine Abnahme der Aktivität beobachtet werden. Aus diesen Ergebnissen kann gefolgert werden, dass es möglich ist, mit Hilfe des erfindungsgemäßen Collagen-Bindungsassays die Aktivität der vWF-spaltenden Protease auf einfache und schnelle Weise zu messen. Dadurch sollte die Diagnose und das Therapiemonitoring von Patienten mit TTP erleichtert werden.In addition, plasmas from a patient with hereditary TTP during therapy with FFP were also examined (FIG. 6). The immunoblotting method is again at the top, and the values obtained with the CBA according to the invention can be seen at the bottom. On the far left is a plasma sample that was taken before therapy. The other samples were taken after a first and a second plasma exchange. Both in the immunoblotting method and in the collagen binding assay according to the invention, an increase in activity after the first exchange, a further increase after the second and then a decrease in activity can be observed. From these results it can be concluded that it is possible to measure the activity of the vWF-cleaving protease in a simple and quick manner with the aid of the collagen binding assay according to the invention. This should facilitate the diagnosis and therapy monitoring of patients with TTP.
Der Vorteil des erfindungsgemäßen Verfahrens ist die Möglichkeit zur quantitativen Auswertung und die einfachere und vor allem kürzere Durchführbarkeit, die in der Akutdiagnostik der lebens- bedrohlichen Krankheitsbilder von entscheidender Bedeutung ist. Eine besonders vorteilhafte Ausführungsform des erfindungsgemäßen Tests verwendet zur quantitativen Bestimmung des Protease- Substrates von von Willebrand Faktor den Collagen-Bindungsassay gemäß AT 403 853.The advantage of the method according to the invention is the possibility of quantitative evaluation and the simpler and, above all, shorter feasibility, which is of crucial importance in the acute diagnosis of the life-threatening clinical pictures. A particularly advantageous embodiment of the test according to the invention uses the collagen binding assay according to AT 403 853 for the quantitative determination of the protease substrate by von Willebrand factor.
Eine weitere Anwendung der vorliegenden Erfindung besteht in der quantitativen Bestimmung der von Willebrand Faktor-spaltenden Protease bei der Reinigung und Charakterisierung als therapeutische Plasmafraktion oder zur Qualitätskontrolle von therapeutisch eingesetzten Vollplasmen. Dies wird durch das nachfolgende Beispiel (siehe Fig. 7) näher erläutert:Another application of the present invention consists in the quantitative determination of the protease splitting von Willebrand factor during purification and characterization as a therapeutic plasma fraction or for quality control of therapeutically used full plasmas. This is explained in more detail by the following example (see Fig. 7):
Die von Willebrand Faktor-spaltende Protease (Multimerase) wurde gemäß AT 404 359 teilweise gereinigt. Zur weiteren chromatographischen Reinigung wurde die Multimerasepr paration auf eine Säule, gefüllt mit Fractogel® TSK AF-Orange (Merck) , aufgetragen und mit einem Puffergradienten von pH 5,5 - pH 8,5 in einem Trispuffer (10 mM Tris, 10 mM Na3.Citrat, 150 mM NaCl) eluiert. In den Fraktionen wurde die UV-Absorption bei 280nm (Gesamtprotein) und die Collagen-Bindungsaktivität eines von Willebrand Faktor-Standards nach erfindungsgemäßer Inkubation mit den Fraktionen gemessen. Der Kehrwert der Collagen-Bindungsaktivität wurde in das Elutionsdiagramm eingetragen und zeigt, dass neben einem Teil der Proteaseaktivität, die sich im Säulendurchlauf (Fraktion 10-20) befindet, der Großteil der Aktivität zwischen Fraktion 33 und 38 eluiert werden konnte. The protease (multimerase) cleaving factor by Willebrand was partially purified according to AT 404 359. For further chromatographic purification the Multimerasepr was paration on a column packed with Fractogel ® TSK AF-Orange (Merck), and eluted with a buffer gradient of pH 5.5 - pH 8.5 in Tris buffer (10 mM Tris, 10 mM Na 3rd citrate, 150 mM NaCl) eluted. In the fractions, the UV absorption at 280 nm (total protein) and the collagen binding activity of a von Willebrand factor standard were measured after incubation with the fractions according to the invention. The reciprocal of the collagen binding activity was entered in the elution diagram and shows that in addition to a part of the protease activity which is in the column run (fraction 10-20), the majority of the activity could be eluted between fractions 33 and 38.

Claims

P a t e n t a n s p r ü ch e: Patent claims:
1. Testkit zur Analytik der von Willebrand Faktor-spaltenden Protease und zur Differentialdiagnostik zwischen Patienten mit thrombotisch thrombozytopenischer Purpura und Patienten mit hämolytisch-urämischem Syndrom, bestehend aus einer von Willebrand Faktor-Standardpräparation, die frei von von Willebrand Faktor- spaltender Aktivität ist, als Substrat für die von Willebrand Faktor-spaltende Aktivität in einer Probe oder im Patientenplasma und einem System zur quantitativen Bestimmung der Bindung von von Willebrand Faktor an Collagen.1. Test kit for the analysis of von Willebrand factor-splitting protease and for differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with haemolytic-uraemic syndrome, consisting of a von Willebrand factor-standard preparation that is free of Willebrand factor-splitting activity, as Substrate for von Willebrand factor-splitting activity in a sample or in patient plasma and a system for the quantitative determination of the binding of von Willebrand factor to collagen.
2. Testkit gemäß Anspruch 1, dadurch gekennzeichnet, dass die quantitative Bestimmung der Collagen-Bindungsaktivität an immobilisiertes avides Collagen erfolgt, welches vorzugsweise an eine Mikrotiterplatte gebunden vorliegt.2. Test kit according to claim 1, characterized in that the quantitative determination of the collagen binding activity is carried out on immobilized avid collagen, which is preferably bound to a microtiter plate.
3. Testkit nach Anspruch 2, dadurch gekennzeichnet, dass das immobilisierte Collagen kovalent gebunden ist .3. Test kit according to claim 2, characterized in that the immobilized collagen is covalently bound.
4. Testkit nach Anspruch 2 oder 3, dadurch gekennzeichnet, dass das avide Collagen ein enzymatisch abgebautes, lösliches, humanes Collagen ist .4. Test kit according to claim 2 or 3, characterized in that the avide collagen is an enzymatically degraded, soluble, human collagen.
5. Testkit gemäß einem der Ansprüche 1 bis 4 , dadurch gekennzeichnet, dass die von Willebrand Faktor-Standardpraparation ein humanes Referenzplasma ist, bei welchem die von Willebrand Faktor-spaltende Proteaseaktivität inaktiviert ist.5. Test kit according to one of claims 1 to 4, characterized in that the von Willebrand factor standard preparation is a human reference plasma in which the protease activity splitting by Willebrand is inactivated.
6. Testkit gemäß einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass die von Willebrand Faktor-Standardpraparation ein rekombinanter von Willebrand Faktor ist.6. Test kit according to one of claims 1 to 4, characterized in that the von Willebrand factor standard preparation is a recombinant von Willebrand factor.
7. Testkit gemäß Anspruch 1 bis 4, dadurch gekennzeichnet, dass die von Willebrand Faktor-Standardpraparation ein von Willebrand Faktor-Dimer ist.7. Test kit according to claim 1 to 4, characterized in that the von Willebrand factor standard preparation is a von Willebrand factor dimer.
8. Verfahren zum Nachweis eines erworbenen oder kongenitalen Mangels der von Willebrand Faktor-spaltenden Protease, dadurch gekennzeichnet, dass eine von Willebrand Faktor-Standardpraparation, die frei von von Willebrand Faktor-spaltender Aktivität ist, als Substrat mit der von Willebrand Faktor-spaltenden Aktivität des Patientenplasmas oder Verdünnungen desselben in Kontakt gebracht wird und nach Inkubation die quantitative Detektion des residualen von Willebrand Faktors durch seine Bindungseigenschaften an Collagen erfolgt . 8. A method for detecting an acquired or congenital deficiency of the protease that splits von Willebrand factor, thereby characterized that a von Willebrand factor standard preparation, which is free of von Willebrand factor-splitting activity, is brought into contact as substrate with the von Willebrand factor-splitting activity of the patient's plasma or dilutions thereof, and after incubation the quantitative detection of the residual von Willebrand Factor due to its binding properties to collagen.
EP00908814A 1999-02-25 2000-02-23 Test kit for analysing factor viii-splitting protease Withdrawn EP1155328A1 (en)

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AT13299 1999-02-25
AT13299 1999-02-25
PCT/AT2000/000049 WO2000050904A1 (en) 1999-02-25 2000-02-23 Test kit for analysing factor viii-splitting protease

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DE20213920U1 (en) 2002-07-03 2003-01-16 Boehm Martina Diagnostic for the detection of the activity of ADAMTS13 that cleaves the Willebrand factor
US7763430B2 (en) * 2003-04-22 2010-07-27 Baxter International Inc. Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies
WO2005008241A1 (en) * 2003-07-07 2005-01-27 University Of North Carolina At Chapel Hill Method and system for detection of von willebrand factor (vwf) multimers
ES2373989T3 (en) 2006-01-31 2012-02-10 Mitsubishi Chemical Medience Corporation PROCEDURE FOR THE DETERMINATION OF THE STATE OF A DISSEMINATED INTRAVASCULAR COAGULATION SYNDROME.
DE102007031708A1 (en) 2007-07-06 2009-01-08 Dade Behring Marburg Gmbh Determination of von Willebrand factor activity in the absence of ristocetin
EP4279607A1 (en) 2022-05-20 2023-11-22 Siemens Healthcare Diagnostics Products GmbH Enzyme-enhanced adamts-13 activity assay

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US6068838A (en) * 1996-04-29 2000-05-30 Baxter Aktiengesellschaft Purified multimerase
AT403853B (en) * 1996-07-04 1998-06-25 Immuno Ag Method for determining the activity of an adhesion protein

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AU756563B2 (en) 2003-01-16
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WO2000050904A8 (en) 2001-04-12

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