AU3133600A

AU3133600A - Test kit for analysing factor viii-splitting protease

Info

Publication number: AU3133600A
Application number: AU31336/00A
Authority: AU
Inventors: Miha Furlan; Helena E. Gerritsen; Bernhard Lammle; Hans-Peter Schwarz; Jurgen Siekmann; Peter Turecek; Katalin Varadi
Original assignee: Baxter AG
Current assignee: Baxter AG
Priority date: 1999-02-25
Filing date: 2000-02-23
Publication date: 2000-09-14
Anticipated expiration: 2020-02-23
Also published as: CA2362483A1; EP1155328A1; WO2000050904A1; WO2000050904A8; AU756563B2

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.

2 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 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 8 42-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.

Claims

10-20), the majority of the activity could be eluted between fraction 33 and 38. Claims 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 voti 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. 5 8. 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.