Heterogeneous coagulation test

The present invention is in the field of coagulation diagnostics and relates to methods for determining the activity of proteolytic clotting factors.

In coagulation diagnostics called global tests to study the functionality of the blood coagulation cascade and so-called Single tests are distinguished for determining the activity of individual coagulation factors. Both for the global tests as well as for the individual tests are known different test formats. With regard to the assay format can be distinguished substantially coagulation tests and chromogenic tests.

In a coagulation test, the patient sample to be examined, which usually consists of plasma is mixed with a coagulation activator, which starts the clotting process. The coagulation process results in the formation of a fibrin clot, which can be measured by photometric methods. The fibrin formation is a measure of the functionality of the blood coagulation cascade. To determine the activity of an individual coagulation factor in a coagulation test, the patient sample to be examined is additionally mixed with a deficient plasma, which all components of the blood coagulation cascade supplemented in addition to the blood coagulation factor to be tested.

In a chromogenic test, the patient sample to be examined, which usually consists of plasma is mixed with a coagulation activator, and with a substrate for a clotting factor. Since this is at most coagulation factors by serine endopeptidases, ie by hydrolases can cleave peptide bonds, peptide substrates are mainly used, which are as specific as possible cleaved from the to be determined, blood clotting factor, and which have a detectable signal group. Preferably, cleavable chromogenic or fluorogenic signal groups are used, which are determined by photometry. In Patent Documents<patcit id="pcit0001" dnum="EP34122A1"><text>EP 34122 A1</text></patcit> and <patcit id="pcit0002" dnum="US4508644A"><text>US 4,508,644</text></patcit> A variety of chromogenic peptide substrates and their use in coagulant diagnostic tests, for. example, for determining the coagulation factors factor IIa (thrombin) and Xa. The document<patcit id="pcit0003" dnum="EP78764A1"><text>EP 78764 A1</text></patcit> describes a chromogenic method for the determination of coagulation factor XIIa.

In particular, using the chromogenic tests can also anticoagulants that inhibit the activity of clotting factors are determined in patient samples. For this purpose, the patient sample to be tested is mixed with an activated clotting factor and a substrate for this clotting factor. The more anticoagulant is contained in the sample, the stronger the activated coagulation factor is inhibited and the less substrate is cleaved.

In these known processes is homogeneous process. Homogeneous assay methods have the advantage that the sample is mixed with the detection reagents to assay and the detection reaction is measured in this assay without requiring additional separation steps, for example to separate the analyte from other sample components, are necessary. However, homogeneous assay methods also have the disadvantage that probenintrinsische substances during the entire test procedure are present, thereby influencing the detection reaction, or may interfere with the measurement of the detection reaction. Patient samples may, for example, in individual cases, contain abnormally high concentrations of one or more intrinsic, ie the body's own substances that turn out when exceeding a tolerable concentration in photometric detection methods as annoying and can affect to a systematic error. Problems are known to be hemolytic, icteric and / or lipemic serum or plasma samples, called HIL samples over abnormally high hemoglobin, bilirubin - feature and / or triglyceride concentrations. Abnormally high concentrations of these interfering substances can be caused by a pathological condition of the patient or from improper sample collection or storage.

The present invention was thus based on the object to provide a method for determining the activity of proteolytic coagulation factors in a sample, which is less susceptible to interference from probenintrinsischen interfering substances.

This object is achieved in that the method comprises the steps of:<ol><li>a. providing and incubating a reaction mixture comprising<ol><li>i. the sample,</li><li>ii. an agent for direct or indirect activation of the proteolytic clotting factor in the sample,</li><li>iii. a cleavable substrate having at least one cleavage site for activated clotting factor,</li><li>iv. a solid phase to which the cleavable substrate is bound or becomes bound during the incubation;</li></ol></li><li>b. Separation of the solid phase; and</li><li>c. Determining the amount of solid phase-bound, uncleaved substrate by incubating the solid phase with a reagent that contains substances that specifically interact with the undigested substrate and produce a measurable signal.</li></ol>

The amount of solid phase-bound, uncleaved substrate is inversely proportional to the activity of the identified proteolytic coagulation factor.

The provision of the reaction mixture always involves contacting the sample, preferably a blood or plasma sample, with an agent for direct or indirect activation of the proteolytic coagulation factor to be determined in the sample and a solid phase. The cleavable substrate having at least one cleavage site for activated clotting factor can be supplied to the reaction mixture in various ways:<ul><li>The cleavable substrate, such as a synthetic peptide or a purified protein which is not bound to a solid phase may be contained in a separate reagent which is added to the reaction mixture. The reaction mixture then contains a solid phase to which the cleavable substrate is bound during the incubation.</li><li>The cleavable substrate, such as a synthetic peptide or a purified protein is already bound to the solid phase and is brought together with the solid phase as a component thereof with the sample and the agent for activating the proteolytic clotting factor in contact.</li><li>The cleavable substrate is a naturally present in the sample, a natural substrate for the activated coagulation factor and is thereby supplied as a constituent of the sample material with the sample to the reaction mixture. The reaction mixture then contains a solid phase to which the cleavable natural substrate binds during incubation.</li></ul>

Fissile substrates which have at least one cleavage site for an activated clotting factor, those skilled well known. A fissile substrate may be a synthetic, recombinant, or bio-engineered molecule or a natural molecule be, which is broken down by the action of the activated clotting factor in two fission products. A fissile substrate may consist entirely or partially of a peptide. Preferably, it comprises at least in the region of a cleavage site peptide portion. Preferably, there is the peptide portion of a cleavable substrate from 3 to about 150 amino acid residues.

In another embodiment, the cleavable substrate consist of a complete protein or a protein fragment. A cleavable substrate can also be a natural substrate of an activated clotting factor. An example of a natural fissionable substrate comprising (thrombin) and plasmin factor V, the cleavage sites for activated protein C, factor Xa, factor IIa. Another example is fibrinogen, which has cleavage sites for factor IIa (thrombin). Yet another example is factor II (prothrombin), which cleavage sites for factor IIa (thrombin), Factor Xa and various snake venoms, such. As having ecarin or textarin.

In one embodiment of the inventive method is the cleavable substrate having at least one cleavage site for the activated coagulation factor, bound to a solid phase.

The term "bound" has a broad meaning and includes, for example, covalent and non-covalent bond, a direct and an indirect binding, adsorption to a surface and inclusion in a depression or cavity, etc. In a covalent bond the cleavable substrate via a chemical bond bound to the solid phase. Examples of a non-covalent bond are surface adsorption, inclusion in cavities or the binding of two specific binding partners. In addition to a direct binding to the solid phase, the cleavable substrate may also be bonded indirectly through specific interaction with other specific binding partners to the solid phase.

In a preferred embodiment, the cleavable substrate a first binding partner A a first binding pair A / B, and the solid phase comprises the binding partner B, and the substrate is bound by binding the binding partner A and B to the solid phase.

In another embodiment of the method according to the invention, the cleavable substrate having at least one cleavage site for activated clotting factor, during the incubation of the reaction mixture bound to the solid phase. For this purpose, the cleavable substrate a first binding partner A a first binding pair A / B, and the solid phase comprises the binding partner B, and the substrate is bound during the incubation the reaction mixture by binding of the binding partners A and B to the solid phase.

Suitable binding pairs A / B are mainly antigen / antibody combinations, wherein the binding partner A is an antigenic epitope of the cleavable substrate. The antigenic epitope may be a natural sequence or a natural Strukturepitop protein or protein fragment, in particular when a natural, contained in the sample substrate is used as the fissile substrate. The antigenic epitope may also be a heterologous sequence or a modified cleavable Strukturepitop substrate. Examples of heterologous sequence or Strukturepitope are FLAG, or HIS or fluorescein tags which are in particular used for labeling peptides or proteins. The solid phase-bound binding partner B should be chosen such that the cleavable substrate can be specifically bound. Preferably, there is the binding partner B from an antibody or an antigen-binding fragment thereof. Particularly preferred binding pairs A / B are FLAG tag / anti-FLAG-tag antibody, HIS-tag / anti-HIS tag antibody and fluorescein / anti-fluorescein antibody.

In a further embodiment From the process of the invention, the cleavable substrate a first binding partner X of a second binding pair X / Y in which said second binding partner Y is interacting with the second binding partner Y of the second binding pair X / Y and associated with a component of a signal-forming system , In this way, uncleaved substrate are detected. The binding partner X is disposed in a region of the substrate, if the substrate is cleaved by the activated proteolytic clotting factor is separated from the solid phase-bound portion of the substrate.

Suitable binding pairs X / Y are, for example, antigen / antibody combinations, wherein the binding partner X an antigenic epitope of the cleavable substrate is. The antigenic epitope may be a natural sequence or a natural Strukturepitop protein or protein fragment, in particular when a natural, contained in the sample substrate is used as the fissile substrate. The antigenic epitope may also be a heterologous sequence or a modified cleavable Strukturepitop substrate. Examples of heterologous sequence or Strukturepitope are FLAG, or HIS or fluorescein tags which are in particular used for labeling peptides or proteins. Other suitable binding pairs X / Y are, for example, biotin / avidin and biotin / streptavidin.

The second binding partner Y is associated with a component of a signal-generating system.

A "signal-generating system" may be one or more components, where there is a detectable label at least one component. A label means any molecule is to be understood that produces even a signal or can induce the production of a signal, such as a fluorescent substance, a radioactive substance, an enzyme or a chemiluminescent substance. The signal may be detected or measured, for example, on the basis of enzyme activity, luminescence, light absorption, light scattering, the emitted electromagnetic or radioactive radiation or a chemical reaction.

A label can itself produce a detectable signal, so that no further components are necessary. Many organic molecules absorb ultraviolet and visible light, which can put these molecules in an excited energy state and emit the absorbed energy in the form of light of a different wavelength than that of the irradiated light. Yet other labels can directly generate a detectable signal, such as radioactive isotopes or dyes.

Yet other labels need for signal generation, further components, ie the signal-producing system includes in such a case, all the components required for the signal formation with an such. As substrates, coenzymes, quenchers, accelerators, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, ions, etc.

Suitable labels are, for example, enzymes including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase, urease and acetylcholinesterase; dyes; fluorescent substances including fluorescein, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-Dimethylaminonapthalen-1-sulfonyl chloride and fluorescent chelates of rare earths; chemiluminescent substances including luminol, isoluminol, acridinium compounds, olefin, enol ether, enamine, aryl vinyl ethers, dioxene, arylimidazole, lucigenin, luciferin and aequorin; Sensitizers including eosin, 9,10-dibromoanthracene, methylene blue, porphyrin, phthalocyanine, chlorophyll, Rose Bengal; Coenzyme; Enzyme substrates; radioactive isotopes including¹²⁵I, ¹³¹I, ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ⁵¹Cr, ⁵⁹Fe, ⁵⁷Co and ⁷⁵Se.

The solid phase to which the cleavable substrate is bound or is bound during the incubation, includes an article which is made of porous and / or nonporous, generally water-insoluble material and can have various shapes, such as for example. The of vessels, tubes, microtiter plates, beads, microparticles, rods, strips, filter or chromatography, etc. In general, the surface of the solid phase is hydrophilic or can be made hydrophilic. The solid phase may consist of different materials, such. As of inorganic and / or organic materials, of synthetic, of naturally occurring and / or modified naturally occurring materials. Examples of solid phase materials are polymers such as cellulose, nitrocellulose, cellulose acetate, polyvinylchloride, polyacrylamide, crosslinked dextran, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate, or nylon; Ceramics, glass, metals, especially precious metals such as gold and silver; magnetite; Mixtures or combinations thereof; etc.

The solid phase may have a coating of one or more layers, eg. As of proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof, for example, to suppress the non-specific binding of sample components to the solid phase or to prevent or to, for example, improvements to achieve with regard to the stability of the suspension of particulate solid phases, the storage stability, the forming stability or resistance to UV light, microbes or other destructive effect agents.

For the direct or indirect activation of the proteolytic clotting factor in the sample, the sample is usually mixed with an agent which causes a direct or indirect activation of the proteolytic clotting factor. Under direct activation is to be understood that an agent is used that activates the proteolytic coagulation factor to be determined directly, regardless of the presence of other clotting factors. Indirect activation is to be understood that an agent is used, the one or more coagulation factors of the blood coagulation cascade is activated, which in turn enable to be examined proteolytic clotting factor. The type of agent depends on which clotting factor is to be determined whether the activity of the clotting factor is to be determined solely or whether the functionality of the blood coagulation cascade or a portion of the blood coagulation cascade (extrinsic or intrinsic pathway) is to be determined on the basis of a clotting factor. Substances and mixtures of different specific substances that enable a direct or indirect activation of proteolytic clotting factors, as are well known to the skilled person and include for example, phospholipids, such as negatively charged phospholipids. Lipoproteins, such as thromboplastin. ., Proteins, such as tissue factor, activated serine proteases, such as factor IIa (thrombin), Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, or activated protein C; Snake venoms, such as Protac® enzyme Ecarin, textarin, noscarin, batroxobin thrombocytin or Russell's viper venom (RVV). Contact activators such. For example, silica, kaolin, ellagic acid or Celite. Further substances which can contain an agent, are, for. Example, buffer substances, salts, detergents, ions, in particular calcium ions and chelating agents.

A "sample" within the meaning of the invention to understand the material containing the detected proteolytic coagulation factor probably. in particular, the term sample comprises human or tiereische body fluids, primarily blood and plasma.

After the reaction mixture that contains the sample, an agent for activation of the proteolytic clotting factor, a cleavable substrate and a solid phase to which the cleavable substrate is bound or becomes bound, is provided, the reaction mixture is incubated for a limited time to to ensure sufficient activation of clotting factor, a sufficient cleavage of the substrate by the activated clotting factor and optionally a sufficient bonding of the cleavable substrate or the cleavage product of the substrate to the solid phase. The term "sufficient" will be understood that the method as a whole allows a quantitative determination of the activity of the clotting factor. The optimum incubation period of a particular test set-up can be determined experimentally.

In a preferred embodiment, the reaction mixture an inhibitor of fibrin also may be added. Under a Fibrinaggregationsinhibitor is a substance to be understood in particular a synthetic oligopeptide having the juxtaposition (polymerization) of the fibrin monomers, which arise by the action of thrombin, inhibited, thereby preventing clot formation in the reaction mixture (see, eg. B.<patcit id="pcit0004" dnum="EP0456152B1"><text>EP 456 152 B1 0</text></patcit>).

After the reaction mixture was incubated, the solid phase and thus the components bound thereto from the remaining constituents of the reaction mixture is separated off. The separation can be done in different ways, eg., By centrifugation, filtration, magnetic separation, or by aspirating the liquid phase of the reaction mixture depending on the nature of the solid phase. After separation of the solid phase, at least one washing step is carried out to completely remove possible residues of the reaction mixture from the solid phase and / or to prepare the solid phase for the subsequent detection reaction. For this, the solid phase is treated with a wash solution, preferably with a buffer solution, incubated and subsequently separated from the washing solution again.

Determination of the amount of solid phase-bound unclipped substrate can be done in different ways depending on the type of detection system used, which interact z. B. by incubating the solid phase with a reagent that contains substances specific to the uncleaved substrate and a measurable generate signal. Preferably, the determination of the amount of solid phase-bound unclipped substrate takes place in that the solid phase is incubated with a detection reagent that contains the binding partner Y of the second binding pair X / Y, which specifically binds to the binding partner X of uncleaved substrate. The binding partner Y can either be directly associated with a signal-generating Kompenente or be associated with a signaling Kompenente.

The amount or strength of the signal is proportional to the amount of solid phase-bound unclipped substrate and thus inversely proportional to the activity of the proteolytic coagulation factor.

The inventive method for the determination of a proteolytic clotting factor is particularly suitable for the determination of proteolytic clotting factor II, factor VII, factor IX, factor X, factor XI, factor XII, or protein C.

The following examples serve to illustrate the present invention and are not intended to be limiting.

figure Description

<ul><li><figref idrefs="f0001"><b>figure 1</b></figref><figref idrefs="f0001">figure 1</figref> shows the absorbance values ​​at 450 nm of samples in which the extrinsic pathway of coagulation (a thromboplastin reagent) was activated by Innovin® and in which the thrombin activity was determined (see Example 1). The samples are standard human plasma (SHP), which fully contains all coagulation factors, and plasmas deficient in factors of the extrinsic (F II, FV, F VII, FX) or the intrinsic coagulation pathway (F VIII, F IX, F XI). Depending on the amount of thrombin in the activated sample, which in turn is the presence of the factors of the extrinsic coagulation pathway dependent on the thrombin-sensitive peptide substrate is cleaved. The more thrombin is present in the sample, the more thrombin substrate is cleaved and the less uncleaved thrombin substrate can be detected. The measured absorbance values ​​are thus inversely proportional to the thrombin activity in the sample or to the activity of the extrinsic coagulation cascade. In the experiment shown, therefore, all the plasmas with lack of factors of the extrinsic coagulation pathway (in particular the FII-deficient plasma) higher absorbance than normal plasma (SHP), whereas plasmas not involved in the extrinsic pathway factors (factor VIII, IX, XI) no signal differences have to SHP. Therefore, it could be clearly demonstrated in plasmas with the test shown a lack of factors of the extrinsic coagulation pathway.</li><li><figref idrefs="f0001"><b>figure 2</b></figref><figref idrefs="f0001">figure 2</figref> shows the absorbance values ​​at 450 nm of samples with different Refludan® concentration, where a defined amounts of thrombin was added (Thr. 1 IU / ml, Thr. 10 IU / ml) and in which the thrombin activity was determined (see Example 2). The more contain some of the direct thrombin inhibitor Refludan® in a sample, the more of the added thrombin is inhibited, the less thrombin is cleaved and the more uncleaved thrombin substrate can be detected. The measured absorbance values ​​are thus proportional to the thrombin-inhibitory activity of the Refludan® in the sample. It became clear that the sensitivity of the method for samples with low therapeutically Refludan® concentrations (up to 1 g / ml) with the addition of a relatively low amount of thrombin (1 IU / ml) is particularly well, while the sensitivity of the method for samples with therapeutically high Refludan® concentrations (1-5 ug / ml) with the addition of a relatively high amount of thrombin (10 IU / ml) is especially good.</li></ul>

Examples

Example 1: Method for determining the thrombin activity

First, microtiter plates (Nunc-Thermo Fisher, Roskilde, Denmark) with a monoclonal FLAG epitope antibody (MAB M2, Sigma Aldrich, Munich, Germany). Subsequently, a seven amino acid residues complete thrombin-specific peptide substrate with the amino acid residues, leucine, valine, proline, arginine, glycine, phenylalanine, glycine in said sequence comprising a FLAG-Tag epitope at the amino terminal end and is biotinylated at the carboxy terminus, incubated with the anti-FLAG antibody solid phase bound and so bound to the solid phase. Pro cavity following reagents were then added in the following order:<ul><li>25 .mu.l of a Fibrinaggregationsinhibitor solution (6 mg / ml of a synthetic oligopeptide having amino acid residues glycine, proline, arginine, proline, alanine in the above sequence); 25 ul of sample; and 25 ul Innovin® (recombinant human tissue factor with synthetic phospholipids, Siemens Healthcare Diagnostics, Marburg, Germany) as a clot.</li></ul>

As samples, standard human plasma (SHP) or coagulation factor-deficient plasmas (factor X, factor V, factor VII, factor VIII, factor IX, factor XI, factor II,) were used.

The reaction mixture was mixed and incubated for 15 minutes at 37 ° C. Then the reaction mixture was aspirated and each well was washed three times with 250 ul wash buffer. For the detection of uncleaved, solid phase-bound thrombin substrate 100 microliters of a streptavidin / peroxidase (POD) were -Konjugatlösung (0.33 ug / ml, Sigma Aldrich) was added and again incubated for 30 minutes at 20-25 ° C into each well , Then, the conjugate solution was aspirated, and each well was washed three times with 250 ul wash buffer. 100 microliters of a buffer solution containing 0.5 g / L TMB (3,3'5,5'-tetramethylbenzidine dihydrochloride) and 0.1 g / L were then in each well of hydrogen peroxide was added and again for 30 minutes at 20-25 ° C incubated. To stop the peroxidase reaction 100 ul was added 0.5 N sulfuric acid to each well, and absorbance at 450 nm minus the reference wavelength 650 nm was determined using a MTP photometer Sunrise® (Tecan Trading AG, Switzerland).

The results are in <figref idrefs="f0001">figure 1</figref> shown.

Example 2: A method for the quantitative determination of direct thrombin inhibitor Refludan®

As described in Example 1, was a seven amino acid residues complete thrombin-specific peptide substrate which has a FLAG-tag epitope at the amino terminal end and being biotinylated at the carboxy-terminal end, bound via an anti-FLAG antibody to the solid phase (microtiter plate).

The following reagents were added in the following order each cavity:<ul><li>50 .mu.l of a Fibrinaggregationsinhibitor solution (3 mg / ml of a synthetic oligopeptide having amino acid residues glycine, proline, arginine, proline, alanine in the above sequence);</li><li>25 ul of sample; and</li><li>25 .mu.l bovine α-thrombin solution (4 or 40 IU of bovine α-thrombin / ml, 10 KIE / ml Aprotinin, 150 mM / L NaCl, 5 mg / ml bovine serum albumin, 10 mg / ml mannitol, 5 ug / ml of hexadimethrine bromide ).</li></ul>

The different concentrations of thrombin, the sensitivity in the different therapeutic areas can be ensured.

As sample standard human plasma (SHP) or normal human citrated plasma samples were used. These were divided into aliquots, and the aliquots 0.2 ug / ml, 1.0 ug / ml and 5 ug / ml Refludan® (lepirudin, recombinant hirudin, CSL Behring GmbH, Marburg were each 0.0 ug / ml,, Germany ) added.

The detection of uncleaved, solid phase-bound thrombin substrate was prepared as described in Example 1, performed, and 650 nm using a MTP photometer Sunrise® (Tecan Trading AG, Switzerland) was the absorbance at 450 nm minus the reference wavelength determined.

The results are in <figref idrefs="f0001">figure 2</figref> shown.