CA2362483A1 - Test kit for analysing factor viii-splitting protease - Google Patents
Test kit for analysing factor viii-splitting protease Download PDFInfo
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- CA2362483A1 CA2362483A1 CA002362483A CA2362483A CA2362483A1 CA 2362483 A1 CA2362483 A1 CA 2362483A1 CA 002362483 A CA002362483 A CA 002362483A CA 2362483 A CA2362483 A CA 2362483A CA 2362483 A1 CA2362483 A1 CA 2362483A1
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- 108091005804 Peptidases Proteins 0.000 title claims abstract description 28
- 239000004365 Protease Substances 0.000 title claims abstract description 28
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 28
- 108010047303 von Willebrand Factor Proteins 0.000 claims abstract description 40
- 102000008186 Collagen Human genes 0.000 claims abstract description 35
- 108010035532 Collagen Proteins 0.000 claims abstract description 35
- 229920001436 collagen Polymers 0.000 claims abstract description 35
- 229960001134 von willebrand factor Drugs 0.000 claims abstract description 33
- 230000000694 effects Effects 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 claims abstract description 7
- 238000003748 differential diagnosis Methods 0.000 claims abstract description 5
- 230000007812 deficiency Effects 0.000 claims abstract description 4
- 206010010356 Congenital anomaly Diseases 0.000 claims abstract description 3
- 230000027455 binding Effects 0.000 claims description 12
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 claims description 8
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000000539 dimer Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 2
- 102100036537 von Willebrand factor Human genes 0.000 abstract 2
- 208000037157 Azotemia Diseases 0.000 abstract 1
- 206010043561 Thrombocytopenic purpura Diseases 0.000 description 7
- 238000000159 protein binding assay Methods 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 230000002950 deficient Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 0 C*=C1CCCCC1 Chemical compound C*=C1CCCCC1 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
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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]
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Engineering & Computer Science (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
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- Biochemistry (AREA)
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- Neurosurgery (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 haemolyt ic- 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 fo r detecting an acquired or congenital deficiency of von Willebrand factor- splitting protease.
Description
TEST KIT FOR ANALYZING FACTOR VIII-SPLITTING PROTEASE
The present invention relates to a test kit and a method for the determination of von Willebrand's factor-splitting protease as well as the application of the method to the differential diagnosis of thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS).
A deficiency of von Willebrand's factor-splitting protease was found in patients with TTP, whereas patients with hemolytic uremic syndrome (HUS) have normal activity of von Willebrand's factor-splitting protease. Except for the various cumbersome methods already described to date (Blood 1996; 87:4223), no method for determining the activity of von Willebrand's factor-splitting protease is currently available. A simple method of determination would facilitate distinguishing between TTP and HUS, and thus allow better monitoring of the therapy of patients with TTP.
It has now surprisingly been found that a test kit consisting of a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as a substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen makes possible for the first time in a simple manner the analysis of the von Willebrand's factor-splitting protease and the differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic uremic syndrome. The von Willebrand's factor standard preparation can in the process be inactivated specifically with regard to the von Willebrand's factor-splitting protease, but a general protease-inactivated von Willebrand's factor standard preparation can also be used.
According to a preferred embodiment of the test kit according to the invention, the collagen binding activity is quantitatively determined on immobilized avid collagen which is preferably bound to a microtiter plate.
In the process, it is favorable for the immobilized collagen to have a covalent bond.
Furthermore, it is advantageous when the avid collagen is a soluble human collagen that is broken down by enzymes.
Preferably, the von Willebrand's factor standard preparation is a human reference plasma in which the von Willebrand's factor-splitting protease activity is inactivated.
According to a further embodiment, it is provided that the von Willebrand's factor standard preparation is a recombinant von Willebrand's factor.
It is also convenient when the von Willebrand's factor standard preparation is a von Willebrand's factor dimer.
Furthermore, the present invention also relates to a method for the detection of an acquired or congenital deficiency of the von Willebrand's factor, in which a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as a substrate is brought into contact with the von Willebrand's factor-splitting activity of the patient plasma or dilutions of same, and after incubation the residual von Willebrand's factor is quantitatively detected through its binding properties to collagen.
The attached figures show the test principle according to the invention and the application to the diagnosis of patient plasma. The agreement of the complicated SDS
agarose gel electrophoretic qualification with subsequent immune blotting with the quantitative method of the collagen binding assay (CBA) according to the invention is also represented in the attached figures.
According to the invention, dilutions of patient and normal plasma were activated with barium, and undiluted vWF substrate was added. As a substrate, a protease-inactivated plasma was used. After 2 h digestion in the presence of urea, the reaction was stopped and the incubation mixture was put on an ELISA plate that had 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 that were bound to the collagen can be detected by means of generally known methods of the state of the art, for example, by staining using peroxidase-labeled antibodies.
The present invention will now be explained in more detail with reference to the attached figures.
Shown are:
Figure l, the principle of the collagen binding test according to the invention, Figure 2, the action of the protease with vWF-splitting activity, Figure 3, the action of the protease with vWF-splitting activity on the vWF
subunit, Figure 4, the test calibration, Figure 5, the determination of the vWF-splitting protease in patients with thrombotic thrombocytopenic purpura, Figure 6, the determination of the vWF-splitting protease in a patient with thrombotic thrombocytopenic purpura during therapeutic treatment, and Figure 7, the quantitative determination of the vWF-splitting protease in purified plasma fractions.
In the test according to the invention (Figure 1), the different affinities of the vWF
multimers of various lengths are exploited. The long multimers have a very high affinity to collagen whereas the small molecules have very little or no affinity.
vWF consists of multimers with different lengths of the same subunit. The subunits are each bound together at the carboxy and amino termini by disulfide bridges (Figure 2). Each subunit can be split at tyrosine 842-methionine 843 by the vWF protease (Figure 3).
Under these conditions a calibration curve was established with different normal plasma dilutions (Figure 4). On the Y axis, the extinction is plotted at 492 nm, and on the X axis, the normal plasma dilutions. It is assumed that a dilution of normal plasma of 1/20 corresponds to 100% activity. The more vWF-splitting protease present in the mixture, the greater the vWF is digested and the less it can bind to collagen, and thus the smaller the extinction.
Presented in Figure 5 are the examinations of two families whose members suffer from hereditary TTP. Shown above is the immune blotting method, and below are the values that were obtained with the collagen binding assay (CBA) according to the invention. On the far left is the propositus, who is homozygous deficient in vWF-splitting protease. Next comes the brother, also homozygous-deficient, the sister who is normal, and both parents who must be homozygous.
Wherever strong digestion of the vWF is indicated, the CBA according to the invention shows a value of 100%; where the substrate was not digested, it was also not possible to detect any activity in the CBA according to the invention. On the right are members of the same family, all of whom are homozygous deficient.
Furthermore, plasma specimens of a patient with hereditary TTP were examined during therapy with FFP (Figure 6). Above, the immune blotting method can again be seen, and below, the values that were obtained with the CBA according to the invention. On the far left, a plasma specimen is listed that was taken before therapy. The other specimens were taken after a first and a second plasma exchange. Both in the immune blotting method as well as in the collagen binding assay according to the invention, an increase of the activity can be observed after the first exchange, a further increase after the second, and afterwards, a reduction of the activity.
From these results it can be concluded that it is possible, using the collagen binding assay according to the invention, to simply and quickly measure the activity of the vWF-splitting protease. Thereby, the diagnosis and therapy monitoring of patients with TTP
can be facilitated.
The advantage of the method according to the invention is the possibility for quantitative assessment, and the ability to perform it in a simpler and quicker manner, which is of critical significance in the acute diagnosis of life-threatening disease 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 of von Willebrand's factor.
Another application of the present invention consists in the quantitative determination of von Willebrand's factor-splitting protease in the purification and characterization as therapeutic plasma fraction, or for quality control of whole plasma used in therapy. This is explained in more detail by means of the following example (see Figure 7).
von Willebrand's factor-splitting protease (multimerase) was purified in part according to AT 404 359. For further chromatographic purification, the multimerase preparation was applied to a column filled with Fractogel~ TSK AF-orange (Merck), and eluted with a buffer gradient of pH 5.5-8.5 in a Tris buffer (10 mM Tris, 10 mM Naycitrate, 150 mM NaCI). In the fractions, the LTV absorption at 280 nm (total protein) and the collagen binding activity of a von Willebrand's factor standard after incubation with the fractions according to the invention were measured. The reciprocal value of the collagen binding activity was entered into the elution diagram and indicates that, aside from a portion of the protease activity that is located i;i the column run-through (fraction 10-20), the majority of the activity could be eluted between fraction 33 and 38.
laims 1. Test kit for the analysis of von Willebrand's factor-splitting protease and for differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic uremic syndrome, consisting of a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen.
The present invention relates to a test kit and a method for the determination of von Willebrand's factor-splitting protease as well as the application of the method to the differential diagnosis of thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS).
A deficiency of von Willebrand's factor-splitting protease was found in patients with TTP, whereas patients with hemolytic uremic syndrome (HUS) have normal activity of von Willebrand's factor-splitting protease. Except for the various cumbersome methods already described to date (Blood 1996; 87:4223), no method for determining the activity of von Willebrand's factor-splitting protease is currently available. A simple method of determination would facilitate distinguishing between TTP and HUS, and thus allow better monitoring of the therapy of patients with TTP.
It has now surprisingly been found that a test kit consisting of a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as a substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen makes possible for the first time in a simple manner the analysis of the von Willebrand's factor-splitting protease and the differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic uremic syndrome. The von Willebrand's factor standard preparation can in the process be inactivated specifically with regard to the von Willebrand's factor-splitting protease, but a general protease-inactivated von Willebrand's factor standard preparation can also be used.
According to a preferred embodiment of the test kit according to the invention, the collagen binding activity is quantitatively determined on immobilized avid collagen which is preferably bound to a microtiter plate.
In the process, it is favorable for the immobilized collagen to have a covalent bond.
Furthermore, it is advantageous when the avid collagen is a soluble human collagen that is broken down by enzymes.
Preferably, the von Willebrand's factor standard preparation is a human reference plasma in which the von Willebrand's factor-splitting protease activity is inactivated.
According to a further embodiment, it is provided that the von Willebrand's factor standard preparation is a recombinant von Willebrand's factor.
It is also convenient when the von Willebrand's factor standard preparation is a von Willebrand's factor dimer.
Furthermore, the present invention also relates to a method for the detection of an acquired or congenital deficiency of the von Willebrand's factor, in which a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as a substrate is brought into contact with the von Willebrand's factor-splitting activity of the patient plasma or dilutions of same, and after incubation the residual von Willebrand's factor is quantitatively detected through its binding properties to collagen.
The attached figures show the test principle according to the invention and the application to the diagnosis of patient plasma. The agreement of the complicated SDS
agarose gel electrophoretic qualification with subsequent immune blotting with the quantitative method of the collagen binding assay (CBA) according to the invention is also represented in the attached figures.
According to the invention, dilutions of patient and normal plasma were activated with barium, and undiluted vWF substrate was added. As a substrate, a protease-inactivated plasma was used. After 2 h digestion in the presence of urea, the reaction was stopped and the incubation mixture was put on an ELISA plate that had 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 that were bound to the collagen can be detected by means of generally known methods of the state of the art, for example, by staining using peroxidase-labeled antibodies.
The present invention will now be explained in more detail with reference to the attached figures.
Shown are:
Figure l, the principle of the collagen binding test according to the invention, Figure 2, the action of the protease with vWF-splitting activity, Figure 3, the action of the protease with vWF-splitting activity on the vWF
subunit, Figure 4, the test calibration, Figure 5, the determination of the vWF-splitting protease in patients with thrombotic thrombocytopenic purpura, Figure 6, the determination of the vWF-splitting protease in a patient with thrombotic thrombocytopenic purpura during therapeutic treatment, and Figure 7, the quantitative determination of the vWF-splitting protease in purified plasma fractions.
In the test according to the invention (Figure 1), the different affinities of the vWF
multimers of various lengths are exploited. The long multimers have a very high affinity to collagen whereas the small molecules have very little or no affinity.
vWF consists of multimers with different lengths of the same subunit. The subunits are each bound together at the carboxy and amino termini by disulfide bridges (Figure 2). Each subunit can be split at tyrosine 842-methionine 843 by the vWF protease (Figure 3).
Under these conditions a calibration curve was established with different normal plasma dilutions (Figure 4). On the Y axis, the extinction is plotted at 492 nm, and on the X axis, the normal plasma dilutions. It is assumed that a dilution of normal plasma of 1/20 corresponds to 100% activity. The more vWF-splitting protease present in the mixture, the greater the vWF is digested and the less it can bind to collagen, and thus the smaller the extinction.
Presented in Figure 5 are the examinations of two families whose members suffer from hereditary TTP. Shown above is the immune blotting method, and below are the values that were obtained with the collagen binding assay (CBA) according to the invention. On the far left is the propositus, who is homozygous deficient in vWF-splitting protease. Next comes the brother, also homozygous-deficient, the sister who is normal, and both parents who must be homozygous.
Wherever strong digestion of the vWF is indicated, the CBA according to the invention shows a value of 100%; where the substrate was not digested, it was also not possible to detect any activity in the CBA according to the invention. On the right are members of the same family, all of whom are homozygous deficient.
Furthermore, plasma specimens of a patient with hereditary TTP were examined during therapy with FFP (Figure 6). Above, the immune blotting method can again be seen, and below, the values that were obtained with the CBA according to the invention. On the far left, a plasma specimen is listed that was taken before therapy. The other specimens were taken after a first and a second plasma exchange. Both in the immune blotting method as well as in the collagen binding assay according to the invention, an increase of the activity can be observed after the first exchange, a further increase after the second, and afterwards, a reduction of the activity.
From these results it can be concluded that it is possible, using the collagen binding assay according to the invention, to simply and quickly measure the activity of the vWF-splitting protease. Thereby, the diagnosis and therapy monitoring of patients with TTP
can be facilitated.
The advantage of the method according to the invention is the possibility for quantitative assessment, and the ability to perform it in a simpler and quicker manner, which is of critical significance in the acute diagnosis of life-threatening disease 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 of von Willebrand's factor.
Another application of the present invention consists in the quantitative determination of von Willebrand's factor-splitting protease in the purification and characterization as therapeutic plasma fraction, or for quality control of whole plasma used in therapy. This is explained in more detail by means of the following example (see Figure 7).
von Willebrand's factor-splitting protease (multimerase) was purified in part according to AT 404 359. For further chromatographic purification, the multimerase preparation was applied to a column filled with Fractogel~ TSK AF-orange (Merck), and eluted with a buffer gradient of pH 5.5-8.5 in a Tris buffer (10 mM Tris, 10 mM Naycitrate, 150 mM NaCI). In the fractions, the LTV absorption at 280 nm (total protein) and the collagen binding activity of a von Willebrand's factor standard after incubation with the fractions according to the invention were measured. The reciprocal value of the collagen binding activity was entered into the elution diagram and indicates that, aside from a portion of the protease activity that is located i;i the column run-through (fraction 10-20), the majority of the activity could be eluted between fraction 33 and 38.
laims 1. Test kit for the analysis of von Willebrand's factor-splitting protease and for differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic uremic syndrome, consisting of a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen.
2. Test kit according to Claim 1, characterized in that the quantitative determination of the collagen binding activity occurs on immobilized avid collagen that is present preferably on a microtiter plate.
3. Test kit according to Claim 2, characterized in that the immobilized collagen has a covalent bond.
4. Test kit according to Claim 2 or 3, characterized in that the avid collagen is soluble human collagen that is broken down by enzymes.
5. Test kit according to one of Claims 1-4, characterized in that the von Willebrand's factor standard preparation is a human reference plasma in which the von Willebrand's factor-splitting protease activity is inactivated.
6. Test kit according to one of Claims 1-4, characterized in that the von Willebrand's factor standard preparation is a recombinant von Willebrand's factor.
7. Test kit according to Claims 1-4, characterized in that the von Willebrand's factor standard preparation is a von Willebrand's factor dimer.
Claims (9)
1. Use of a test kit containing a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen.
2. Use according to Claim 1 for the analysis of von Willebrand's factor-splitting protease and for differential diagnosis between patients with thrombotic thrombocytopenic purpura and patients with hemolytic uremic syndrome.
3. Test kit containing a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity as substrate for the von Willebrand's factor-splitting activity in a specimen or in the patient plasma, and a system for the quantitative determination of the binding of von Willebrand's factor to collagen.
4. Test kit according to Claim 3, characterized in that the quantitative determination of the collagen binding activity occurs on immobilized avid collagen that is present preferably on a microtiter plate.
5. Test kit according to Claim 3 or 4, characterized in that the immobilized collagen has a covalent bond.
6. Test kit according to one of Claims 3-5, characterized in that the avid collagen is soluble human collagen that is broken down by enzymes.
7. Test kit according to one of Claims 2-6, characterized in that the von Willebrand's factor standard preparation is a recombinant von Willebrand's factor.
8. Test kit according to Claims 2-6, characterized in that the von Willebrand's factor standard preparation is a von Willebrand's factor dimer.
9. Method for the detection of an acquired or congenital deficiency of von Willebrand's factor-splitting protease characterized in that a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity, as a substrate, is brought into contact with the von Willebrand's factor-splitting activity of the patient plasma or dilutions of same, and after incubation, the residual von Willebrand's factor is quantitatively detected by its binding properties to collagen.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT13299 | 1999-02-25 | ||
| ATGM132/99 | 1999-02-25 | ||
| PCT/AT2000/000049 WO2000050904A1 (en) | 1999-02-25 | 2000-02-23 | Test kit for analysing factor viii-splitting protease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2362483A1 true CA2362483A1 (en) | 2000-08-31 |
Family
ID=3482389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002362483A Abandoned CA2362483A1 (en) | 1999-02-25 | 2000-02-23 | Test kit for analysing factor viii-splitting protease |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1155328A1 (en) |
| AU (1) | AU756563B2 (en) |
| CA (1) | CA2362483A1 (en) |
| WO (1) | WO2000050904A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8759018B2 (en) | 2006-01-31 | 2014-06-24 | Mitsubishi Chemical Medience Corporation | Method for determining treatment of disseminated intravascular coagulation |
| US8932820B2 (en) | 2007-07-06 | 2015-01-13 | Siemens Healthcare Diagnostics Products Gmbh | Methods and kits for determining von Willebrand factor activity in the absence of ristocetin and for determining the activity of ADAMTS-13 protease |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE20213920U1 (en) | 2002-07-03 | 2003-01-16 | Böhm, Martina, 60439 Frankfurt | 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 |
| JP5112692B2 (en) * | 2003-07-07 | 2013-01-09 | ユニヴァーシティ・オヴ・ノース・キャロライナ・アト・チャペル・ヒル | Method and system for detecting von Willebrand factor (VWF) multimers |
| EP4279607A1 (en) | 2022-05-20 | 2023-11-22 | Siemens Healthcare Diagnostics Products GmbH | Enzyme-enhanced adamts-13 activity assay |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2000
- 2000-02-23 AU AU31336/00A patent/AU756563B2/en not_active Ceased
- 2000-02-23 EP EP00908814A patent/EP1155328A1/en not_active Withdrawn
- 2000-02-23 CA CA002362483A patent/CA2362483A1/en not_active Abandoned
- 2000-02-23 WO PCT/AT2000/000049 patent/WO2000050904A1/en not_active Ceased
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8759018B2 (en) | 2006-01-31 | 2014-06-24 | Mitsubishi Chemical Medience Corporation | Method for determining treatment of disseminated intravascular coagulation |
| US8932820B2 (en) | 2007-07-06 | 2015-01-13 | Siemens Healthcare Diagnostics Products Gmbh | Methods and kits for determining von Willebrand factor activity in the absence of ristocetin and for determining the activity of ADAMTS-13 protease |
| US9222942B2 (en) | 2007-07-06 | 2015-12-29 | Siemens Healthcare Diagnostics Products Gmbh | Methods and kits for determining von willebrand factor activity in the absence of ristocetin |
| US9869683B2 (en) | 2007-07-06 | 2018-01-16 | Siemens Healthcare Diagnostics Products Gmbh | Methods and kits for determining von Willebrand factor activity in the absence of ristocetin |
| US10591490B2 (en) | 2007-07-06 | 2020-03-17 | Siemens Healthcare Diagnostics Products Gmbh | Methods and kits for determining von willebrand factor activity in the absence of ristocetin |
Also Published As
| Publication number | Publication date |
|---|---|
| AU756563B2 (en) | 2003-01-16 |
| WO2000050904A1 (en) | 2000-08-31 |
| WO2000050904A8 (en) | 2001-04-12 |
| AU3133600A (en) | 2000-09-14 |
| EP1155328A1 (en) | 2001-11-21 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |