Heterogeneous coagulation test

Heterogeneous coagulation test.

The present invention is in the field of coagulation diagnosis and refers to methods for determining the activity of blood coagulation proteolytic factors.

In the diagnosis of coagulation, the so-called global tests are differentiated to investigate the functionality of the blood coagulation cascade and the so-called individual tests to determine the activity of individual blood coagulation factors. Different assay formats are known for both global and individual trials. With respect to the test format, coagulation tests and chromogenic tests are essentially differentiated.

In the coagulation test, the sample of the patient to be investigated that usually consists of plasma is mixed with a coagulation activator which starts the coagulation process. The coagulation process leads to the formation of a fibrin clot that can be measured with the help of photometric methods. The rate of 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 investigated is mixed with a deficient plasma that provides all the components of the blood coagulation cascade, except the blood coagulation factor to be tested. .

In a chromogenic assay, the patient sample to be investigated, which is usually composed of plasma, is mixed with a coagulation activator and a substrate for a coagulation factor. Since the majority of coagulation factors are serine-endopeptidases, that is hydrolases that can dissociate peptide bonds, predominantly peptide substrates are used that are dissociated as specifically as possible by the blood coagulation factor to be determined and which have a group of detectable signal. More preferably, dissociable chromogenic or fluorogenic signal groups that are determined photometrically are used. A large number of chromogenic peptide substrates and their use in diagnostic coagulation assays are described in EP 34122 A1 and US 4,508,644, for example to determine the coagulation factors factor IIa (thrombin) and Xa. A chromogenic process for determining coagulation factor XIIa is described in EP 78764 A1.

Anticoagulants that inhibit the activity of blood coagulation factors can also be determined in patient samples, mainly with the help of chromogenic assays. For this purpose, the patient sample to be examined is mixed with an activated coagulation factor and with a substrate for this coagulation factor. The more anticoagulant is contained in the sample, the stronger the activated coagulation factor is inhibited and the less substrate dissociates.

These known methods are homogeneous methods. Homogeneous test methods have the advantage that the sample is mixed with the detection reagents to form a test mixture and the detection reaction is measured in this test mixture without the need for additional separation steps, for example, to separate the analyte of the other sample components. However, homogeneous test methods also have the disadvantage that intrinsic substances are present in the sample during the entire test method and, as a result, may affect the detection reaction or may interfere with the measurement of the detection reaction. . Patient samples may contain, for example in individual cases, abnormally high concentrations of one or more intrinsic substances, that is, endogenous, which prove to be interfering by exceeding a tolerable concentration in photometric detection methods and can produce a systematic error. It is known that the problems are caused by hemolytic, icteric and / or lipemic serum or plasma samples, the so-called HIL samples that have abnormal high concentrations of hemoglobin, bilirubin and / or triglycerides. Abnormal high concentrations of these interfering substances may be caused by a pathological condition of the patient or by improper collection or storage of the sample.

The fundamental object of the present invention was to provide a method for determining the activity of proteolytic coagulation factors in a sample which was less susceptible to interference by a harmful substance intrinsic to the sample.

This object is achieved, according to the invention, by the fact that the method comprises the following steps:

<dl><dt>to.</dt><dd> provide and incubate a reaction mixture containing </dd></dl>

<dl><dt>i.</dt><dd> the sample </dd></dl>

ii. an agent for the direct or indirect activation of the proteolytic coagulation factor in the sample,

iii. a dissociable substrate that has at least one cutoff site for the activated coagulation factor,

<dl><dt>iv.</dt><dd> a solid phase to which the dissociable substrate is bound or bound during incubation; </dd></dl>

<dl><dt>b.</dt><dd> Separate the solid phase; and</dd></dl>

<dl><dt>c.</dt><dd> Determine the amount of substrate bound to the solid phase, not dissociated by incubation, with a reagent containing substances that interact specifically with the non-dissociated substrate and generate a measurable signal. </dd></dl>

The amount of substrate bound to the solid phase, not dissociated, has a proportion inversely proportional to the activity of the proteolytic coagulation factor to be determined.

Providing the reaction mixture always comprises contacting the sample, preferably a blood or plasma sample, with an agent for the direct or indirect activation of the proteolytic coagulation factor to be determined in the sample and with a solid phase. The dissociable substrate that has at least one cut-off site for the activated coagulation factor can be introduced into the reaction mixture in different ways:

<dl><dt>-</dt><dd> The dissociable substrate, for example a synthetic peptide or a purified protein, which is not bound to a solid phase, may be contained in a separate reagent that is added to the reaction mixture. The reaction mixture then contains a solid phase to which the dissociable substrate is bound during incubation.</dd></dl>

<dl><dt>-</dt><dd>The dissociable substrate, for example a synthetic peptide or a purified protein, is already bound to the solid phase and contacts, together with the solid phase as components thereof, with the sample and the agent for the activation of the factor of proteolytic coagulation </dd></dl>

<dl><dt>-</dt><dd> The dissociable substrate is a natural substrate, naturally contained in the sample, for the activated coagulation factor and thus introduced into the reaction mixture as a component of the sample material with the sample. The reaction mixture then contains a solid phase to which the natural dissociable substrate is bound during incubation.</dd></dl>

Dissociable substrates that have at least one cut-off site for an activated coagulation factor are well known to the person skilled in the art. A dissociable substrate can be a synthetic molecule, produced recombinantly or by biotechnology or it can be a natural molecule that decomposes into two dissociation products by the action of the activated coagulation factor. A dissociable substrate can be composed entirely or partially of a peptide. This preferably comprises a peptide fraction at least in the region of the cutting site. The peptide fraction of a dissociable substrate is preferably composed of 3 to about 150 amino acid residues.

In another embodiment, the dissociable substrate may be composed of a complete protein or a protein fragment. A dissociable substrate can also be a natural substrate of an activated coagulation factor. An example of a natural dissociable substrate is factor V, which has cut sites for activated protein C, factor Xa, factor IIa (thrombin) and plasmin. Another example is fibrinogen that has cut sites for factor IIa (thrombin). A further example is factor II (prothrombin), which has cut-off sites for factor IIa (thrombin), factor Xa and various snake venoms such as, for example, ecarin or textarin.

In an embodiment of the method of the invention, the dissociable substrate, which has at least one cut-off site for the coagulation factor, is linked to a solid phase.

The term "bound" can be broadly understood and comprises, for example, a covalent and a non-covalent bond, a direct and an indirect bond, adsorption to a surface and inclusion in a cavity or hollow space, etc. in the case of a covalent bond, the dissociable substrate is linked by a chemical bond to the solid phase. Examples of a non-covalent bond are adsorption to a surface, inclusion in hollow spaces or the bonding of two specific binding participants. In addition to a direct link to the solid phase, the dissociable substrate can also be indirectly linked to the solid phase by specific interaction with other binding participants.

In a preferred embodiment, the dissociable substrate has a first link A participant of a first A / B link pair, and the solid phase has link B participant, and the substrate is linked by link A link participant A and B to the solid phase.

In another embodiment of the method of the invention the dissociable substrate, which has at least one cut-off site for the activated coagulation factor, is bound to the solid phase during incubation of the reaction mixture. For this, the dissociable substrate has a first link A participant of a first A / B link pair, and the solid phase has link B participant, and the substrate is bound during incubation of the reaction mixture via the link of the participants of link A and B to the solid phase.

Suitable A / B binding pairs are, first and foremost, antigen / antibody combinations, in which case the binding partner A is an antigenic epitope of the dissociable substrate. The antigenic epitope can be a sequence or natural structure epitope of a natural protein or a protein fragment, especially when a natural substrate contained in the sample is used as a dissociable substrate. The antigenic epitope can also be an epitope of sequence or heterologous structure of a modified dissociable substrate. Examples of heterologous sequence or structure epitopes are FLAG-, or HIS- or fluorescein-Tags, which are primarily used to label peptides or proteins. The B-link participant bound to the solid phase can be selected such that the dissociable substrate can be specifically linked. The B-binding participant is preferably composed of an antibody or a fragment thereof that forms an antigen. Particularly preferred A / B binding pairs are FLAG-Tag / anti-FLAG-Tag-antibody, HIS-Tag / anti-HIS-Tag-antibody and fluorescein / anti-fluorescein-antibody.

In another embodiment of the method of the invention, the dissociable substrate has a first X link participant of a second X / Y link pair, which interacts with the second Y link participant of the second X / Y link pair and in this case, the second link participant Y is associated with a component of a signal forming system. In this way the undissociated substrate can be detected. The linker X is arranged in a region of the substrate that is separated from the region of the substrate that is linked to the solid phase, provided that the substrate is dissociated by the activated proteolytic coagulation factor.

Suitable X / Y binding pairs are, for example, antigen / antibody combinations, in which case the X binding participant is an antigenic epitope of the dissociable substrate. The antigenic epitope can be a sequence or natural structure epitope of a natural protein or protein fragment, especially when a natural substrate contained in the sample is used as a dissociable substrate. The antigenic epitope can also be an epitope of sequence or heterologous structure of a modified dissociable substrate. Examples of heterologous sequence or structure epitopes are FLAG or HIS or fluorescein-tags (tags), which are mainly used for peptide or protein labeling. Other suitable X / Y binding pairs are for example biotin / avidin and biotin / streptavidin.

The second linker Y is associated with a component of a signal forming system.

A "signal forming system" can be one or more components, in which case the component is a detectable label. A tag can be understood as any molecule that produces a signal by itself or that can induce the production of a signal such as, for example, a fluorescent substance. A radioactive substance, an enzyme or a chemiluminescent substance. The signal can be detected or measured, for example, by enzymatic activity, luminescence, light absorption, light scattering, emitted or radioactive electromagnetic radiation or a chemical reaction

A tag itself is capable of generating a detectable signal so that other components are needed. Many organic molecules absorb ultraviolet and visible light, whereby these molecules can reach an excited energy state and the absorbed energy is emitted in the form of light of another wavelength than the excitation light. Other tags such as radioactive isotopes or dyes can directly generate a detectable signal.

For signal generation, other tags need other components, that is, the signal producing system includes in such a case all the components necessary for signal formation such as substrates, coenzymes, quencher (suffocators), accelerators, enzymes additional substances that react with enzyme products, catalysts, activators, cofactors, inhibitors, ions, etc.

Suitable labels are, for example, enzymes, including horseradish peroxidase, alkaline phosphatases, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase, urease and acetylcholinesterase; dyes; fluorescent substances, including fluoresceinaisothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonylchloride and fluorescent rare earth chelates; Chemiluminescent substances including luminol, isoluminol, acridinium compounds, olefin, enol ethers, enamine, arylvinyl ethers, dioxen, arylimidazole, lucigenin, luciferin and aequorin; sensitizer (sensitizer) including eosin, 9,10-dibromoanthracene, methylene blue, porphyrin, phthalocyanine, chlorophyll, rose bengal; coenzymes; enzyme substrates; Radioactive isotopes that include 125I, 131I, 14C, 3H, 32P, 33P, 35S, 51Cr, 59Fe, 57Co and 75Se.

The solid phase to which the dissociable substrate is bound or to which it is bound during incubation, contains an object that is composed of porous and / or non-porous material, usually hydro-insoluble and can have the most diverse forms such as, for example , container shapes, tubes, microtiter plates, spheres, microparticles, sticks, strips, filter paper or chromatography. Usually the surface of the solid phase is hydrophilic or can be made hydrophilic. The solid phase may consist of the most diverse materials, such as inorganic and / or organic materials, made of synthetic materials, of natural origin and / or of modified natural origin. Examples of solid phase materials are polymers such as, for example, cellulose, nitrocellulose, cellulose acetate, polyvinylchloride, polyacrylamide, crosslinked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; ceramics, glass, metals, mainly noble metals such as gold and silver; magnetite; mixtures or combinations thereof; etc.

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

For direct or indirect activation of the proteolytic coagulation factor in the sample, the sample is usually mixed with an agent that produces a direct or indirect activation of the proteolytic coagulation factor. By direct activation it is understood that an agent that directly activates the proteolytic coagulation factor to be determined is used, regardless of the presence of other coagulation factors. Indirect activation means that an agent that activates one or more blood coagulation factors of the blood coagulation cascade is used, which in turn activates the proteolytic coagulation factor to be investigated. The type of agent depends on which coagulation factor should be determined, if the activity of the coagulation factor should be determined alone or if the functionality of the blood coagulation cascade or a partial region of the blood coagulation cascade should be determined (via extrinsic or intrinsic) by a coagulation factor. Substances and specific mixtures of different substances that enable direct or indirect activation of proteolytic coagulation factors are well known to the person skilled in the art and comprise, for example, phospholipids such as, for example, negatively charged phospholipids; lipoproteins such as thromboplastin; proteins such as, for example, tissue factor, activated serine proteases such as, for example, factor IIa (thrombin), factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa or activated protein C; snake venoms such as, for example, PROTAC® enzyme, ecarina, textarina, noscarina, batroxobina, trombocitina or Russell's Viper Venom (RVV); contact activators such as silica, kaolin, ellagic acid or celite. Other substances that an agent may contain are, for example, pH buffer substances, salts, detergents, ions, mainly calcium ions and chelate-forming agents.

A "sample" within the meaning of the invention is understood as the material that supposedly contains the proteolytic coagulation factor to be detected. The term "sample" mainly comprises human or animal body fluids, particularly blood and plasma.

After the reaction mixture containing the sample has been prepared, an agent for the activation of the proteolytic coagulation factor, a dissociable substrate and a solid phase to which the dissociable substrate is bound or bound, the reaction mixture is incubate for a limited time in order to ensure sufficient activation of the clotting factor, a sufficient dissociation of the substrate by the activated coagulation factor and optionally a sufficient bond of the dissociable substrate and the dissociation product of the substrate to the solid phase. By the term "sufficient" it is understood that the method makes possible together a quantitative determination of the activity of the coagulation factor. The optimal incubation duration of a given assay assembly can be determined experimentally.

In a preferred embodiment, a fibrin aggregation inhibitor can be added to the reaction mixture. By a fibrin aggregation inhibitor is meant a substance, mainly a synthetic oligopeptide that inhibits the addition of (fibr) to each other the fibrin monomers, which arise from the action of thrombin and thereby prevents a clot formation in the reaction mixture (see, for example EP 0 456 152 B1).

After the reaction mixture is incubated, the solid phase is separated, and with it the components linked thereto, from the other components of the reaction mixture. The separation can be carried out differently according to the type of the solid phase, for example by centrifugation, filtration, magnetic separation or by suction of the liquid phase of the reaction mixture. After separation of the solid phase, at least one washing step can be carried out in order to remove the residues from the reaction mixture as completely as possible from the solid phase and / or to prepare the subsequent detection reaction. For this, the solid phase is incubated with a wash solution, preferably with a pH buffer solution, and then separated again with the wash solution.

The determination of the amount of undissociated substrate, bound to the solid phase can be carried out differently according to the type of the detection system used, for example by incubation of the solid phase with a reagent containing substances that interact specifically with the substrate not dissociated and generate a measurable signal. The determination of the amount of non-dissociated substrate, bound to the solid phase is preferably carried out by incubating the solid phase with a detection reagent containing the Y link participant of the second X / Y link pair, which specifically binds to the link X participant of the undissociated substrate. The Y link participant can be directly associated with a signaling component or be associated with a signaling component.

The amount or intensity of the signal has a proportional relationship with the amount of undissociated substrate, bound to the solid phase and thereby inversely proportional to the activity of the proteolytic coagulation factor.

The method of the invention for determining a proteolytic coagulation factor is mainly suitable for determining proteolytic coagulation factors 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 should not be construed as a restriction.

Description of figures

Figure 1

Figure 1 shows the extinction values ​​at 450 nm of samples in which the extrinsic coagulation pathway was activated by Innovin® (a thromboplastin reagent) and in which thrombin activity was determined (see example 1). The samples are a Standard Human Plasma (SHP), which contains in its entire range all coagulation factors, as well as plasmas with deficiency of extrinsic pathway factors (F II, FV, F VII, FX) and the pathway intrinsic (F VIII, F IX, F XI). Depending on the amount of thrombin in the activated sample which, in turn, depends on the presence of extrinsic coagulation pathway factors, the thrombin-sensitive peptide substrate is dissociated. The more thrombin is contained in the sample, the more thrombin substrate is dissociated and the less undissociated thrombin substrate can be detected. The measured extinction values ​​are thus inversely proportional to the thrombin activity in the sample and to the activity of the extrinsic coagulation cascade. Therefore, in the experiment shown all plasmas with deficiency of extrinsic pathway factors (mainly FII deficiency plasma) extinction values ​​higher than normal plasma (SHP); on the contrary, plasmas with factors not involved in the extrinsic coagulation pathway (factor VIII, IX, XI) have no signal differences with SHP. Therefore, a deficiency of extrinsic coagulation pathway factors in plasmas could be uniquely detected with the assay shown.

Figure 2

Figure 2 shows the extinction values ​​at 450 nm of samples with different concentration of Refludan® to which a defined amount of thrombin was added (Thr. 1 IU / ml, Thr. 10 IU / ml) and in which it was determined thrombin activity (see example 2). The more the direct refludan® thrombin inhibitor is contained in a sample, the more the thrombin added is inhibited, the less the thrombin substrate is dissociated and the greater the undissociated thrombin substrate can be detected. The extinction values ​​measured are in proportion to the thrombin inhibitory activity Refludan® in the sample. In this case it was obvious that the sensitivity of the method for samples with therapeutically low concentrations of Refludan ® (up to 1 g / ml) when adding a comparatively low amount of thrombin (1 IU / ml) is particularly good, while the sensitivity of the method for samples with therapeutically high concentrations of Refludan® (1-5 g / ml) when adding a comparatively high amount of thrombin (10 IU / ml) is particularly good.

Examples

Example 1: Method to determine thrombin activity

First plates and microtiter (Nunc-Thermofisher, Roskilde, Denmark) were coated with a FLAG-epitope-specific monoclonal antibody (MAK M2, Sigma Aldrich, Munich, Germany). A thrombin-specific peptide substrate comprising seven amino acid residues was then incubated, with the amino acid residues leucine, valine, proline, arginine, glycine, phenylalanine, glycine in the aforementioned sequence, which has an epitope at the aminoterminal end FLAG-Tag and is biotinylated at the carboxy terminal end, with the anti-FLAG-antibody bound to the solid phase and thus bound to the solid phase. The following reagents were added in the following sequence per cavity:

25 1 of a fibrin aggregation inhibitor solution (6 mg / ml of a synthetic oligopeptide with the remains of amino acids glycine, proline, arginine, proline, alanine in the sequence mentioned); 25 l of sample; and 25 l of Innovin® (Tissue Recombinant Human Factor with Synthetic Phospholipids, Siemens Healthcare Diagnostics, Marburg, Germany) as a coagulation activator.

Standard Human Plasma (SHP) and coagulation factor deficient plasmas (factor II, factor X, factor V, factor VII, factor VIII, factor IX, factor XI) were used as samples.

The reaction mixture was mixed and incubated for 15 minutes at 37 ° C. Then the reaction mixture was sucked and each cavity was washed three times respectively with 250 l of wash buffer. To detect undissociated thrombin substrate, bound to the solid phase in each cavity 100 l of a streptavidin / peroxidase (POD) conjugate solution (0.33 g / ml, Sigma Aldrich) was added and in turn incubated for 30 minutes at 20-25 ° C. The conjugate solution was then suctioned and each cavity was washed three times respectively with 250 wash buffer. Then in each cavity 100 l of a buffer solution with 0.5 g / L of TMB (3,3,5,5'-tetramethylbenzidine dihydrochloride) and 0.1 g / L of hydrogen peroxide were added and incubated turn for 30 minutes to 20-25 ° C To stop the peroxidase reaction to each cavity 100 l of 0.5 N sulfuric acid, and the extinction at 450 nm with respect to the reference wavelength 650 nm was determined by means of a MTP Sunrise® photometer (Tecan Trading AG , Switzerland).

The results are represented in figure 1.

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

As described in example 1, a thrombin-specific peptide substrate comprising seven amino acid residues, which has a FLAG-Tag epitope and is biotinylated at the aminoterminal end, is linked to the solid phase (microtiter plate ) through an anti-FLAG-antibody.

5 The following reagents were added by cavity in the following sequence:

fifty 1 of a fibrin aggregation inhibitor solution (3 mg / ml of a synthetic oligopeptide with the amino acid residues glycine, proline, arginine, proline, alanine in the sequence mentioned);

25 l of sample; and

25 l of bovine α-thrombin solution (4 or 40 IU of bovine α-thrombin / ml, 10 KIE / ml of aprotinin, 150 mM / L of 10 NaCl, 5 mg / ml bovine serum albumin, 10 mg / ml mannitol, 5 g / ml hexadimetrinbromide). The different concentrations of thrombin can guarantee sensitivity in different therapeutic fields. Standard Human Plasma (SHP) and normal human citrate plasma samples are used as samples. These they made aliquots and 0.0 g / ml, 0.2 g / ml, 1.0 g / ml and 5 g / ml of aliquots were added respectively

fifteen Refludan® (lepirudin, recombinant hirudin, CSL Behring GmbH, Marburg, Germany).

The detection of non-dissociated thrombin substrate, bound to the solid phase, was performed as described in example 1, and the extinction at 450 nm with respect to the reference wavelength of 650 nm was determined by means of a photometer MTP Sunrise® (Tecan Trading AG, Switzerland).

The results are represented in figure 2.