Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • 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

Definitions

• the invention relates to a diagnostic method for the detection of the VWF-splitting ADAMTS-13 activity in blood plasma and other media.
• thrombocytopenic purpura is a disease in which the classic symptoms of thrombocytopenia, microangiopathic, hemolytic anemia, neurological symptoms, renal dysfunction and fever are observed.
• Unusually large multimers of von Willebrand factor (VWF) are found in the plasma of TTP patients and are considered to be the cause of the formation of thrombi rich in VWF and platelets.
• the von Willebrand factor is released by endothelial cells in the form of large multimers, which are split in normal plasma by the interaction of a reductase and a metalloprotease.
• ADAMTS-13 a disintegrin and metalloprotease with thrombospondin motifs
• ADAMTS-13 activity is typically measured by incubating a urea or guanidium hydrochloride treated VWF sample with diluted plasma at low ionic strength.
• the proteolysis is detected by means of a multimer analysis using SDS agarose gel electrophoresis or by fragment analysis using SDS polyacrylamide gel electrophoresis and subsequent immunoblotting, ie the detection of the proteins on a cellulose membrane using the antigen-antibody reaction (4, 5).
• the ADAMTS-13-catalyzed breakdown of von Willebrand factor can also be measured by measuring the collagen Binding activity of the VWF (WO-A 00/50904) or by a specific, two-sided ELISA detection (6) can be determined.
• a recombinant, monomeric VWF which has been labeled with a green fluorescent protein at the N-terminus for determining proteolysis has also recently been described (7).
• the conventional electrophoretic methods can only be carried out in specialized research laboratories, since the tests require special laboratory equipment and the necessary expertise.
• the collagen binding test (WO-A 00/50904) and the specific ELISA (6) for the detection of the proteolytic activity of ADAMTS-13 simplify the ADAMTS-13 activity determination, but can also only be carried out in laboratories that use the appropriate equipment and the necessary know-how.
• the two-sided ELISA also requires specific monoclonal antibodies, which are only available to a few laboratories because they are not commercially available. The task was therefore to develop a simple method for the determination of ADAMTS-13 activity. This new method should allow reliable and timely quantification of ADAMTS-13 activity in blood plasma and other body fluids (e.g. blood serum, saliva) and other media.
• the new method should determine the area quickly and as far as possible Enable ADAMTS 13 activity.
• the timely determination of ADAMTS-13 activity is also essential due to alternative therapy options.
• the method should enable rapid detection of an inhibitor against ADAMTS-13 or the determination of the inhibitor titer, since this results in various therapeutic options (eg rituximab or immune adsorption).
• the procedure should also allow a differentiation between congenital and acquired TTP.
• a timely and routine determination of the ADAMTS-13 activity is the prerequisite for the use of the potentially available, recombinant ADAMTS-13 (W0242441).
• the method should not only allow prompt diagnosis and monitoring of the therapy of TTP patients, but also allow reliable quantification of ADAMTS-13 activity in any media. Since ADAMTS-13 is an important regulator for the VWF and therefore an important factor for hemostasis, the novel method should be applicable in various studies on the importance of ADAMTS-13-catalyzed proteolysis of the VWF in healthy people and patients with different diseases.
• the ADAMTS-13-catalyzed proteolysis of the VWF can be detected by its ability to aggregate platelets.
• the splitting of the VWF by ADAMTS-13 leads to a shortening of the VWF multimers.
• the ability of the VWF to aggregate platelets is largely determined by the length of the multimers. The larger the VWF multimers, the higher their platelet binding capacity. This connection was used in the method according to the invention to determine the ADAMTS-13 activity.
• the invention accordingly relates to a diagnostic method for determining the VWF-splitting activity of ADAMTS-13 in a test medium, the test medium being treated with 0.5 to 5 U / ml of an ADAMTS-13-free von Willebrand factor (VWF) and after incubation the ADAMTS-13 activity is determined by the decrease in the VWF-mediated aggregation of platelets.
• Test medium in the sense of the invention are all body fluids such as B. blood plasma, blood serum, saliva and cerebrospinal fluid and other test media such as. B. cell culture supernatant or cell extracts.
• the test medium is mixed with an ADAMTS-13-free VWF, which is referred to below as the VWF substrate.
• a highly purified, plasmatic VWF which has the multimer pattern typical of normal plasma and is free of endogenous ADAMTS-13 activity is preferably used as the VWF substrate.
• various other VWF substrates which do not have ADAMTS-13 activity such as e.g. B. recombined VWF, VWF from cell culture supernatant or plasmatic VWF, in which the ADAMTS-13 activity has been irreversibly inhibited.
• FIG. 1A The typical multimer pattern of a highly purified, plasmatic VWF is shown in FIG. 1A in the first column.
• the following 7 columns show the time-dependent loss of the high molecular weight VWF multimers by the ADAMTS-13-catalyzed proteolysis.
• a sample was taken from a test batch after the incubation times (0.5-22 h) indicated in FIG. 1A and the reaction was stopped by adding EDTA.
• the samples were subsequently separated by SDS agarose gel electrophoresis in accordance with customary standard methods and then displayed by subsequent immunoblotting.
• a sample for determining the functional activity of the VWF was taken from the reaction mixture after the corresponding incubation times.
• ristocetin cofactor activity The RCo activity was determined using the commercially available BC von Willebrand reagent from Dade Behring (Marburg, Germany). The RCo activity of the corresponding samples measured in this way is indicated under the columns of immunoblotting in FIG. 1A. It can be seen that the time-dependent ADAMTS-13-catalyzed loss of the high molecular weight VWF multimers leads to a significant loss of RCo activity from 357% at the start of the reaction to 13% after 22 hours of incubation. FIG. 1B again illustrates this time-dependent ADAMTS-13 catalyzed loss of RCo activity of the added VWF substrate in the reaction medium.
• the procedure according to the invention is such that 0.5-5 U / ml, preferably 1-3 U / ml, of an ADAMTS-13-free von Willebrand factor (VWF) with the diluted test medium, e.g. B. Blood plasma added.
• the test medium should be diluted so that the endogenous VWF is negligible, ie it does not trigger any detectable platelet aggregation.
• ADAMTS-13-free VWF and the diluted test medium are then incubated for a sufficiently long time, preferably 0.1-24 hours, particularly preferably 6-15 hours. According to the studies available, it even appears possible to reduce the incubation time to 0.5-6 minutes, preferably 1 to 3 minutes.
• This incubation is expediently carried out in a low-molar reaction buffer (eg 5-20 mmol / l TRIS-HCl, pH8) and preferably in the presence of divalent cations, eg. B. Ba 2+ or Ca 2+ , a serine protease inhibitor, e.g. B. PefaBlocSC, and urea.
• divalent cations eg. B. Ba 2+ or Ca 2+
• a serine protease inhibitor e.g. B. PefaBlocSC
• urea urea.
• the preferred concentration of divalent cations in the reaction medium is 5 to 15 mmol / l, particularly preferably 7-9 mmol / l.
• the preferred concentration of Pefabloc in the reaction medium is 0.5 to 6.5 mmol / l, particularly preferably 0.75 mmol / l.
• the preferred concentration of urea in the reaction medium is 0.5 to 3 mol / l, particularly preferably 1.5 mol / l.
• the remaining ability of the VWF substrate to aggregate platelets is determined. This can be measured, for example, using the ristocetin cofactor activity (which describes the VWF-mediated aggregation of platelets in the presence of ristocetin), preferably using the commercially available BC von Willebrand reagent from Dade Behring (Marburg, Germany), and is preferably carried out automatically on one coagulation analyzer. For this purpose, a certain amount of platelets and ristocetin is added to the reaction medium.
• the preferred concentration of platelets in the test mixture is 1,000,000 to 100,000 platelets / ⁇ l, particularly preferably 500,000 to 200,000 platelets / ⁇ l.
• the preferred concentration of ristocetin in the test mixture is 0.5 to 3 mg / ml, particularly preferably 1 to 2 mg / ml.
• the VWF substrate in the reaction medium causes in Presence of ristocetin an aggregation of platelets. The aggregation that takes place reduces the turbidity of the reaction mixture. The ability of the VWF substrate for thrombocyte aggregation can thus be quantified by measuring the optical density.
• the ristocetin cofactor activity of the VWF substrate in the reaction medium determined in this way depends on the ADAMTS-13 activity in the test medium.
• ADAMTS-13 activity in the test medium the more the VWF substrate added is broken down and loses its ability to aggregate platelets.
• Human normal plasma is used for calibration, which is diluted with varying amounts of inactivated human normal plasma (the activity of ADAMTS-13 is inactivated). The inactivation can take place, for example, by means of heating. This relationship is shown in Figure 2.
• the y-axis shows the ability of the corresponding sample to aggregate platelets in the presence of ristocetin, measured in a decrease in absorbance in 90 seconds (mU / 1.5 min).
• Dilution of the normal plasma leads to a limited loss of ADAMTS-13 activity in the test sample, the 1:21 dilution being defined as 100%.
• the content of ADAMTS-13 in the test medium determines the ristocetin cofactor activity of the substrate in the reaction medium.
• the VWF's ristocetin cofactor activity is usually determined in plasma (DE 199 64 109 A 1) and is a common and quick test in any well-equipped routine
• the successful application of the test in the method according to the invention is particularly surprising, since here the RCo activity of a VWF substrate is measured in a very non-physiological reaction medium with a low buffer concentration and in the presence of a serine protease inhibitor and urea to determine the RCo activity in a given plasma sample Plasma (or the corresponding test medium) diluted to such an extent that the endogenous VWF cannot trigger a detectable aggregation of the platelets, ie the endogenous RCo activity is ⁇ 2.
• an exogenous ADAMTS-13-free VWF is added to the plasma thus diluted, which is degraded by the ADAMTS-13 activity in the diluted plasma sample.
• the degradation of the exogenous VWF substrate is quantified by determining the RCo activity.
• the method according to the invention can be described schematically as the action of the ADAMTS-13 present in the test medium on an ADAMTS-13-free VWF substrate and thus a change in the VWF activity (shortening of the VWF multimers).
• Measuring the changed VWF activity by determining the VWF-mediated aggregation of platelets in the presence of ristocetin. Equating the extent of the reduction in VWF activity with the ADAMTS-13 activity of the test medium.
• the ADAMTS-13-free VWF substrate can also be attached to a solid phase, e.g. B. a micro titer plate, or a microparticle, e.g. B. be associated via a specific binding partner.
• a solid phase e.g. B. a micro titer plate, or a microparticle, e.g. B. be associated via a specific binding partner.
• solid phase in the sense of this invention includes an object which consists of porous and / or non-porous, usually water-insoluble material and can have a wide variety of shapes, such as B. vessel, tube, microtitration plate, sphere, microparticles, rods, strips, filter or chromatography paper, etc.
• the surface of the solid phase is hydrophilic or can be made hydrophilic.
• the solid phase can consist of a wide variety of materials such as. B. from inorganic and / or from organic materials, from synthetic, from naturally occurring and / or from modified naturally occurring materials. Examples of solid phase materials are polymers such as. B.
• cellulose nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; ceramics; Glass; Metals, especially precious metals such as gold and silver; magnetite; Mixtures or combinations thereof; etc.
• Cells, liposomes or phospholipid vesicles are also included in the term solid phase.
• the solid phase may have a coating of one or more layers, e.g. B. from proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof, for example to suppress or prevent the non-specific binding of sample components to the solid phase or to achieve improvements in terms of the suspension stability of particulate solid phases, the storage stability, for example shaping stability or resistance to UV light, microbes or other destructive agents.
• layers e.g. B. from proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof, for example to suppress or prevent the non-specific binding of sample components to the solid phase or to achieve improvements in terms of the suspension stability of particulate solid phases, the storage stability, for example shaping stability or resistance to UV light, microbes or other destructive agents.
• microparticles is understood to mean particles which have an approximate diameter of at least 20 nm and not more than 20 ⁇ m, usually between 40 nm and 10 ⁇ m, preferably between 0.1 and 10 ⁇ m, particularly preferably between 0.1 and 5 ⁇ m, very particularly preferably between 0.15 and 2 ⁇ m.
• the microparticles can be regular or irregular in shape. They can represent spheres, spheroids, spheres with more or less large cavities or pores.
• the microparticles can consist of organic, inorganic material or a mixture or a combination of both. They can consist of a porous or non-porous, a swellable or non-swellable material.
• the microparticles can have any density, but preference is given to particles with a density which approximates the density of the water, such as about 0.7 to about 1.5 g / ml.
• the preferred microparticles can be suspended in aqueous solutions and are stable in suspension for as long as possible. They may be translucent, partially translucent, or opaque.
• the microparticles can consist of several layers, such as the so-called "core-and-shell" particles with a core and one or more enveloping layers.
• the term microparticles includes, for example, dye crystals, metal sols, silica particles, glass particles, magnetic particles, particles with chemiluminescent and / or fluorescent substances, polymer particles, oil drops, lipid particles, dextran and protein aggregates.
• Preferred microparticles are particles which can be suspended in aqueous solutions and consist of water-insoluble polymer material, in particular of substituted polyethylenes.
• Latex particles are very particularly preferred.
• reactive groups on their surface such as carboxyl, amino or aldehyde groups, which form a covalent bond e.g. B. allow specific binding partners to the latex particles.
• the production of latex particles is described for example in EP 0080614, EP 0227054 and EP 0246 446.
• a microparticle may have a coating of one or more layers, e.g. B. from proteins, carbohydrates, biopolymers, organic polymers or mixtures thereof, for example to suppress or prevent the non-specific binding of sample components to the particle surface or to achieve, for example, improvements in the suspension stability of the microparticles, the shaping stability or resistance to UV light, microbes or other destructive agents. So this coating can in particular from protein layers or polymer layers such. B. cyclodextrins, dextrans, hydrogels, albumin or polyalbumin exist, which, for. B. covalently or adsorptively applied to the microparticles.
• B. from proteins, carbohydrates, biopolymers, organic polymers or mixtures thereof, for example to suppress or prevent the non-specific binding of sample components to the particle surface or to achieve, for example, improvements in the suspension stability of the microparticles, the shaping stability or resistance to UV light, microbes or other destructive agents. So this coating can in particular from protein layers or polymer layers such. B. cycl
• association encompasses, for example, a covalent and a non-covalent bond, a direct and an indirect bond, the adsorption on a surface and the inclusion in a depression or a cavity etc.
• a covalent bond between two molecules when at least one atomic nucleus of the one molecule shares electrons with at least one atomic nucleus of the second molecule.
• a non-covalent bond are surface adsorption, inclusion in cavities or the binding of two specific binding partners.
• the object to be bound can also be bound indirectly to the solid phase via specific interaction with specific binding partners.
• a “specific binding partner” means a member of a specific binding pair.
• the members of a specific binding pair are two molecules, each of which has at least one structure that is complementary to a structure of the other molecule, the two molecules being able to bind via a binding of the complementary structures.
• the term molecule also includes molecular complexes such as e.g. B. enzymes consisting of apo and coenzyme, proteins consisting of several subunits, lipoproteins consisting of protein and lipids etc.
• Specific binding partners can occur naturally, but also z. B. substances produced by means of chemical synthesis, microbiological techniques and / or genetic engineering processes. To illustrate the term specific binding partner, but not to be understood as a limitation, e.g.
• thyroxine-binding globulin thyroxine-binding globulin, steroid-binding proteins, antibodies, antigens, haptens, enzymes, lectins, nucleic acids, repressors, oligo- and polynucleotides, protein A, protein G, avidin, streptavidin, biotin, grain component C1q, nucleic acid-binding proteins, etc.
• Specific binding pairs are, for example: antibody-antigen, antibody-hapten, operator-repressor, nuclease nucleotide, biotin-avidin, lectin-polysaccharide, steroid-steroid-binding protein, active substance-active substance receptor, hormone -Hormone receptor, enzyme substrate, IgG protein A, complementary oligo- or polynucleotides, etc.
• this can also take place under the influence of shear forces.
• B. be carried out in a flowing medium.
• the invention further relates to a diagnostic kit containing an ADAMTS-13-free VWF substrate, platelets and optionally ristocetin.
• the invention further relates to the use of a reagent for the detection of VWF activity for the detection of the activity of ADAMTS-13.
• the method according to the invention was compared in detail with the immunoblotting method previously used.
• the diagnostic method according to the invention was used to determine the ADAMTS-13 activity in 14 TTP patients during acute attacks, in the course of therapy and in remission, also in 80 healthy subjects and in 23 patients with thrombocytopenia and / or hemolysis and in 14 patients with the Antiphospholipid syndrome (APS) used.
• APS Antiphospholipid syndrome
• Citrate-added plasma samples were obtained from 14 patients in 22 acute TTP attacks before plasma exchange with freshly frozen plasma. The initial diagnosis was based on clinical symptoms (especially neurological logical disorders) and laboratory findings such as severe thrombocytopenia and the detection of microangiopathic, hemolytic anemia. Plasma samples in remission were also obtained from 11 patients. Blood could also be obtained from 10 patients during plasma exchange therapy. Plasma samples from 23 patients with acute thrombocytopenia and / or hemolysis, 14 patients with antiphospholipid syndrome and 80 healthy volunteers were also analyzed. Platelet-poor plasma was produced by centrifugation at 4 ° C. and 2500 g for 40 minutes. The supernatant was then stored at -20 ° C until use.
• Plasma samples were diluted 1:21 with 5 mM Tris-HCL buffer, pH 8, the 12.5 mM barium chloride (BaCI 2 ) and 1 mM PefaBloc SC, an inhibitor of serum proteases (AppliChem GmbH, Darmstadt, Germany) and then Incubated for 5 minutes at 37 ° C to activate the protease.
• a cleaned VWF Concendre de Facteur Willebrand Humain Tres Haute Purite, LFB France
• the concentrate which was free of detectable ADAMTS-13 activity, was reconstituted with aqua ad iniectabilia to a concentration of 100 U / ml, divided and stored at -20 ° C. until use.
• the substrate was thawed, diluted 1:20 with 5 M urea in 5 mM Tris-HCl, pH 8, and incubated for 5 minutes at room temperature. Then 100 ⁇ l of the substrate solution were added to 210 ⁇ l of diluted plasma and left to act at 37 ° C. overnight. The residual ristocetin cofactor activity of the VWF substrate added in the reaction medium was then determined using the commercial, so-called BC von Willebrand reagent from Dade Behring (Marburg, Germany). The ADAMTS-13 activity of a plasma mixture obtained from 80 apparently healthy, adult persons in a dilution of 1:21 was defined as 100%.
• Plasma samples were diluted 1: 5 with 5 mM Tris buffer, pH 8 including 12.5 mM BaCI 2 and 1 mM PefaBloc SC, and the activation and action of the protease were carried out in the same manner as described for the method according to the invention.
• the reaction was stopped by adding disodium EDTA to a final concentration of 23.5 mM.
• the multimer analysis was carried out by SDS electrophoresis on a 1.4% strength agarose (Seakem HGT from Biozym Diagnostics, Hess, Oldenburg, Germany) and an anti-VWF antibody conjugated with peroxidase (P0226 from Dako- Glostrup, Denmark).
• Serial dilutions of normal plasma were tested for quantitative determination, as already described above.
• the results of an exemplary test by the immunoblotting method are shown in FIG. 3.
• various dilutions (1: 5 to 1: 160) of a normal human plasma were mixed with VWF substrate.
• Columns 8-10 show plasma samples from Pa- served with TTP.
• the ADAMTS-13 activity of the test samples is estimated from the calibration in columns 1-7.
• the method according to the invention can also be used for the determination of the inhibitory activity against ADAMTS-13.
• the determination of the inhibitory activity against ADAMTS-13 is of particular clinical importance for the distinction between congenital and acquired TTP. Furthermore, the detection of an inhibitor or the determination of the inhibitor titer is essential for the decision about alternative therapy options (e.g. rituximab or immune adsorption).
• alternative therapy options e.g. rituximab or immune adsorption.
• the ADAMTS-13 activity in the mixtures was determined using the method according to the invention or the previously usual immunoblotting method.
• the ADAMTS- 13 activity of the test sample was divided by the activity of the researchareasmi- 'and multiplied by 100 to determine the residual activity of ADAMTS-13 activity.
• samples with a residual activity of 25 to 75% were selected and the amount of the inhibitor as described for inhibitors of blood coagulation factor VIII was determined (8).
• a sample that caused 50% inhibition of normal ADAMTS-13 activity contained 1 U / ml inhibitor by definition.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Hematology (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Physics & Mathematics (AREA)
• Zoology (AREA)
• Urology & Nephrology (AREA)
• Microbiology (AREA)
• Wood Science & Technology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Pathology (AREA)
• Biophysics (AREA)
• General Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• Food Science & Technology (AREA)
• Cell Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=7714815

Family Applications (1)

Country Status (5)

Families Citing this family (6)

Family Cites Families (7)

• 2002
  • 2002-09-10 DE DE20213920U patent/DE20213920U1/de not_active Expired - Lifetime
• 2003
  • 2003-07-03 EP EP03762597A patent/EP1520035A2/de not_active Withdrawn
  • 2003-07-03 JP JP2004518675A patent/JP2005535315A/ja active Pending
  • 2003-07-03 US US10/519,824 patent/US7291479B2/en not_active Expired - Fee Related
  • 2003-07-03 WO PCT/EP2003/007080 patent/WO2004005451A2/de active Application Filing

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events