AU756563B2 - Test kit for analysing factor VIII-splitting protease - Google Patents
Test kit for analysing factor VIII-splitting protease Download PDFInfo
- Publication number
- AU756563B2 AU756563B2 AU31336/00A AU3133600A AU756563B2 AU 756563 B2 AU756563 B2 AU 756563B2 AU 31336/00 A AU31336/00 A AU 31336/00A AU 3133600 A AU3133600 A AU 3133600A AU 756563 B2 AU756563 B2 AU 756563B2
- Authority
- AU
- Australia
- Prior art keywords
- factor
- von willebrand
- collagen
- splitting
- activity
- 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.)
- Ceased
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Classifications
-
- 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]
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 1, 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 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 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 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 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 ofvon 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 Na3-citrate, 150 mM NaCI). In the fractions, the UV 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 in the column run-through (fraction 10-20), the majority of the activity could be eluted between fraction 33 and 38.
Claims (4)
1. A method for the detection and quantitative determination of a von Willebrand's factor splitting protease in a specimen or in a patient's plasma, comprising subjecting the specimen or plasma to a von Willebrand's factor standard preparation that is free of von Willebrand's factor-splitting activity, and assaying for von Willebrand's factor-splitting protease activity by quantitatively determining the binding of von Willebrand's factor to collagen.
2. The method of claim 1 wherein the specimen or plasma is derived from patients with thrombotic thrombocytopenic purpura and patients with haemolytic uremic syndrome, and is used for differential diagnosis between such patients.
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 a patient's 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, characterised in that the quantitative determination of the collagen binding activity occurs on immobilised avid collagen that is present preferably in a microtiter plate. Test kit according to claim 3 or claim 4, characterised in that the immobilised collagen has a covalent bond. A RAZ 6. Test kit according to one of claims 3-5, characterised in that the avid t collagen is soluble human collagen that is broken down by enzymes. A 28/10102
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 (2)
| Publication Number | Publication Date |
|---|---|
| AU3133600A AU3133600A (en) | 2000-09-14 |
| AU756563B2 true AU756563B2 (en) | 2003-01-16 |
Family
ID=3482389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU31336/00A Ceased AU756563B2 (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) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| AU2004258113B2 (en) * | 2003-07-07 | 2009-11-05 | University Of North Carolina At Chapel Hill | Method and system for detection of von Willebrand factor (vWF) multimers |
| AU2007210643B2 (en) | 2006-01-31 | 2013-09-19 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for determination of condition of 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 |
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 CA CA002362483A patent/CA2362483A1/en not_active Abandoned
- 2000-02-23 WO PCT/AT2000/000049 patent/WO2000050904A1/en not_active Application Discontinuation
- 2000-02-23 EP EP00908814A patent/EP1155328A1/en not_active Withdrawn
- 2000-02-23 AU AU31336/00A patent/AU756563B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000050904A1 (en) | 2000-08-31 |
| CA2362483A1 (en) | 2000-08-31 |
| EP1155328A1 (en) | 2001-11-21 |
| AU3133600A (en) | 2000-09-14 |
| WO2000050904A8 (en) | 2001-04-12 |
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| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) |