Components, instruments, and methods for testing blood coagulation.

Abstract

The present invention provides blood coagulation testing components and other devices that have excellent testing capabilities and can be easily used for blood coagulation testing. [Solution] A blood coagulation testing member 10 comprising a first substrate 11 for contacting a subject containing blood components, and a second substrate 12 that contacts the first substrate 11 (contact portion 13) and contains a quenchable thrombin activity detection substrate and a coagulation activating substance.

Description

[Technical Field]

[0001] This invention relates to a component for testing blood coagulation; a device for testing blood coagulation using the component; and a testing method using the component. [Background technology]

[0002] Blood coagulation tests are blood tests that examine the ability to form blood clots (thrombi). Abnormally increased coagulation ability can lead to thrombosis such as myocardial infarction and cerebral infarction, while abnormally decreased coagulation ability can cause massive bleeding and, in the worst case, death. Actual coagulation tests are performed by adding a substance or reagent that initiates coagulation to the blood and measuring the time it takes for the blood to coagulate. Currently, these tests are widely used to diagnose thrombosis and bleeding, and to assess the effectiveness of blood-thinning drugs (antithrombotic drugs) in patients taking them.

[0003] Generally, coagulation tests require complex and highly specialized procedures, and are therefore performed centrally in facilities such as hospital clinical laboratories, testing centers, and hygiene testing laboratories staffed with clinical laboratory technologists. Consequently, it has been difficult to perform coagulation tests individually in small medical facilities such as hospitals or clinics, at the bedside within hospitals, in emergency medical settings such as intensive care units, or even at home.

[0004] In recent years, there has been a growing need for a testing environment that allows patients taking antithrombotic drugs such as new-generation anticoagulants (DOACs) to routinely monitor the effects and side effects of their medications. Specifically, there is a need for point-of-care rapid coagulation tests that can be easily performed at the patient's regular clinic or at home. Such coagulation tests are easily portable because the equipment is small and lightweight. In addition to clinics and homes, these tests are also expected to be useful in detecting patients with or at high risk of developing the three major thrombosis conditions: cerebral infarction, myocardial infarction, and venous thrombosis (economy class syndrome), in evacuation centers during natural disasters such as earthquakes, in vehicles, at health checkup centers, medical examination facilities, and elderly care facilities.

[0005] Thrombin, one of the blood coagulation factors, plays a central role in thrombus formation. Thrombin is produced by a series of chemical reactions called the blood coagulation cascade (Figure 1). It is thought that thrombin production occurs when a small amount of thrombin produced in the initial reaction amplifies the thrombin formation reaction through a feedback reaction, thereby producing the thrombin necessary for thrombus formation. Under normal conditions, thrombin production is appropriately controlled by the presence of coagulation inhibitors (e.g., tissue factor coagulation pathway inhibitors, TFPIs).

[0006] However, when arteriosclerosis, cancer, and infections develop, thrombin production increases abnormally, resulting in thrombosis, which clogs blood vessels. Conversely, if thrombin production decreases for any reason, it causes bleeding symptoms that are difficult to stop. Therefore, the thrombin production test, which measures thrombin production in the blood, is being established not only as a diagnostic tool for various diseases involving thrombosis, but also as a new coagulation test for early prediction and forecasting of diseases (Non-patent documents 1-4, Patent documents 1-2).

[0007] The thrombin production test is a coagulation test that quantifies the amount of thrombin activity produced (generated) by adding a coagulation activating or initiating reagent, such as tissue factor (TF), to a blood sample and allowing it to react for a certain period of time. Since the measured amount of thrombin is positively correlated with the coagulation ability and thrombus formation ability of the test blood sample, it can serve as a coagulation indicator in the diagnosis of thrombosis and bleeding (Patent Document 1).

[0008] Generally, thrombin activity is quantified using artificially produced low-molecular-weight peptide-based thrombin substrates. The thrombin substrates used are labeled with chromogenic dyes, fluorescent substances, or electrochemically active substances. In tests using substrates labeled with dyes or fluorescent substances, the substrate is cleaved and decomposed by the action of thrombin, releasing the dye or fluorescent substance and causing a change in color or fluorescence intensity. Thrombin is quantified by monitoring this change with spectroscopic optical detection devices such as absorbance or fluorescence plate readers. In tests using electrochemical substrates, the substrate is similarly cleaved and decomposed by the action of thrombin, and the resulting electrochemically active substance is converted into an electrochemical signal by oxidation and / or reduction on an electrode. This signal is then detected by a sensor to quantify thrombin (Patent Document 3).

[0009] Some electrochemical substrate-based thrombin production testing methods are already in practical use and are employed as point-of-care coagulation tests for monitoring warfarin, an antithrombotic drug. For example, the Expressia Stride™ (Siemens) sensor generates electrically active 4-amino-2-chloroferol (ACP) from thrombin within the sensor. Subsequently, a weak voltage is applied to the electrode, causing the ACP to oxidize at the anode while other components are reduced at the cathode, generating a weak electric current. Thrombin is measured by detecting this current. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Patent No. 6883899 [Patent Document 2] Patent No. 7152084 [Patent Document 3] Patent No. 4662596 [Non-patent literature]

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0012] While the usefulness of a test means incorporating an electrochemical substrate has been recognized, due to the use of a complex reaction mechanism, there are problems such as high costs for materials including electrodes and manufacturing costs, and difficulty in selling at a low price. In order to introduce a thrombin production test for point-of-care into daily tests at home, clinics, etc., a simple test means is required while maintaining the test function.

[0013] Fluorescent thrombin substrates can be produced relatively inexpensively by peptide synthesis, and since their test principle is not complex and they do not require special electrodes, etc., the manufacturing costs may be kept low (Patent Documents 1, 2). However, there was room for further study on test means using fluorescent thrombin substrates.

[0014] Under such circumstances, an object of the present invention is to provide a member for testing blood coagulation that has an excellent test function and can be easily used for testing blood coagulation.

Means for Solving the Problems

[0015] As a result of intensive studies to solve the above problems, the present inventor has found that the following invention meets the above object, and has arrived at the present invention. That is, the present invention relates to the following inventions.

[0016] <1> A test member for blood coagulation, comprising a first substrate for contacting a subject containing blood components, and a second substrate that contacts the first substrate and contains a substrate for detecting thrombin activity with extinction and a coagulation activator substance. <2> The test member according to <1>, wherein the substrate for detecting thrombin activity with extinction is an oligopeptide derivative that emits fluorescence when separated into a fragment containing a fluorescent reagent and a fragment containing a quenching reagent upon contact with thrombin. <3> The test member according to <2>, wherein the coagulation activator substance is one or more selected from the group consisting of tissue factor (TF), active blood coagulation factors, and snake venom-derived activator substances that cause thrombin generation by reacting with blood. <4> The test member for blood coagulation according to any one of <1> to <3> is disposed, A test instrument for blood coagulation, comprising a supply unit for supplying the subject to the first substrate and an observation unit for observing the reaction state of the second substrate. <5> A method for testing blood coagulation using a test member having a first substrate for contacting a subject containing blood components, and a second substrate that contacts the first substrate and contains a substrate for detecting thrombin activity with extinction and a coagulation activator substance, the method comprising: supplying a subject to the first substrate; irradiating the second substrate with excitation light; detecting the light emission state of the second substrate; and comparing with an index for evaluating the state of blood coagulation based on the light emission state to determine blood coagulation.

Advantages of the Invention

[0017] According to the present invention, blood coagulation can be easily tested with an excellent test function.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram showing a blood coagulation cascade reaction mechanism. [Figure 2] It is a schematic diagram of the test member according to the present invention. [Figure 3] This is a schematic diagram of the inspection device according to the present invention. [Figure 4] This is a schematic diagram of the inspection method according to the present invention. [Figure 5] This is a structural example illustrating the mechanism of action of the quenchable thrombin activity detection substrate used in the present invention. [Figure 6] This is a graph showing the thrombin activity in the example. [Figure 7] This graph shows the fluorescence intensity of Nma in the example. [Figure 8] This graph shows the substrate hydrolysis activity of the examples. [Figure 9] This graph shows the amount of thrombin produced in the plasma of the example. [Figure 10] This graph shows the amount of thrombin produced in the plasma of the example. [Figure 11] This graph shows the amount of thrombin produced in the plasma of the example. [Modes for carrying out the invention]

[0019] The embodiments of the present invention will be described in detail below, but the description of the constituent elements described below is just one example (representative example) of an embodiment of the present invention, and the present invention is not limited to the following unless its gist is changed. In this specification, when the expression "~" is used, it is used to mean an expression that includes the numbers before and after it.

[0020] [Inspection component of the present invention] The present invention provides a blood coagulation testing component comprising a first substrate for contacting a subject containing blood components, and a second substrate that contacts the first substrate and contains a quenchable thrombin activity detection substrate and a coagulation activating substance.

[0021] [Inspection method of the present invention] The present invention provides a method for testing blood coagulation using a testing member comprising a first substrate for contacting a subject containing blood components, and a second substrate that contacts the first substrate and contains a quenchable thrombin activity detection substrate and a coagulation activating substance, the method comprising the steps of supplying a subject to the first substrate, irradiating the second substrate with excitation light, detecting the luminescence state of the second substrate, and determining blood coagulation by comparing the luminescence state with an index for evaluating the state of blood coagulation based on the luminescence state.

[0022] Furthermore, in this application, the inspection method of the present invention can also be performed using the inspection members, etc. of the present invention, and the corresponding configurations in this application can be used interchangeably.

[0023] The present invention relates to a test for obtaining information to diagnose various coagulation disorders caused by abnormalities in blood coagulation function, and more particularly to a test component that utilizes a luminescent reagent for point-of-care blood coagulation testing to monitor the effectiveness and side effects of therapeutic drugs and procedures for these disorders.

[0024] Figure 1 shows the blood coagulation cascade reaction mechanism. This figure illustrates the coagulation reaction scheme and the thrombin production mechanism. In the conventional coagulation reaction scheme, the prothrombinase complex and thrombin are thought to be produced through three consecutive coagulation reaction phases: initiation, amplification, and propagation. A small amount of thrombin produced via the TF-FVIIa coagulation pathway in the initiation phase triggers positive feedback in the amplification phase and further increases thrombin production in the propagation phase.

[0025] Figure 2 is a schematic diagram of an inspection member according to the present invention. The inspection member 10 has a first base material 11 and a second base material 12. The first base material 11 and the second base material 12 are in contact at a contact portion 13. Figure 3 is a schematic diagram of an inspection device according to the present invention. The inspection device 20 can house the inspection member 10 and be positioned in a predetermined location. The inspection device 20 has a supply section 21 and an observation section 22.

[0026] [Inspection component (10)] The testing component is a component used to evaluate blood coagulation. The testing component can be in the form of a test strip or similar material. The testing component can be used for testing by replacing it for each sample with simple operation.

[0027] [Subject] This thrombin production test can be used to examine indicators of various diseases involving blood coagulation activity. The test specimen can be a biological sample collected from a living body, such as blood, plasma, or urine. The sample volume can be approximately 20-200 μL, preferably 20-100 μL, more preferably 20-60 μL, and particularly preferably 30-50 μL. These specimens may be used in their collected state, or they may be mixed with other chemicals, filtered, or centrifuged as appropriate. They may also be stored by refrigeration or freezing.

[0028] The testing components of the present invention can be used for mammals that have a blood coagulation reaction mechanism. A typical application is the testing of human blood coagulation. Furthermore, they can be used for animals such as monkeys, dogs, cats, cows, pigs, horses, rats, mice, and guinea pigs. They can also be used for pet mammals, livestock animals, and laboratory animals.

[0029] [First substrate (11)] The first substrate is the part to which the sample is supplied. The first substrate has a portion that comes into contact with the second substrate. The sample supplied to the first substrate moves to the second substrate and reacts with reagents, etc., supported on the second substrate. This first substrate serves as a support for a sample pad on which a sample, such as a blood sample like whole blood or plasma, is dropped.

[0030] [Second base material (12)] The second substrate contains a quenchable thrombin activity detection substrate and a coagulation activating substance. The second substrate also has a portion that contacts the first substrate. This second substrate acts like a reagent pad support in the measurement area. This reagent pad support is impregnated with the quenchable thrombin activity detection substrate in a dry form and the coagulation activating substance, and when integrated with the first substrate, it becomes a luminescent biosensor.

[0031] The inspection components of the present invention utilize a quenchable thrombin activity detection substrate that exhibits dramatically improved specificity for thrombin.

[0032] The components of a blood sample dropped onto the first substrate move to the second substrate via capillary flow, and the amount of thrombin produced by their reaction with coagulation activators in the reagent pad is measured. By moving the components of the blood sample through the first substrate, the components move in a gradual dispersion manner. In particular, components such as coagulation inhibitors that react with coagulation activators move quickly on the substrate due to their small molecular size compared to the pore size of the substrate, and selectively move to the second substrate to react. On the other hand, blood and other samples contain contaminants such as red blood cells and white blood cells, which are impurities in blood coagulation tests. These contaminants may react with coagulation activators or affect fluorescence and quenching, but due to their large molecular size, their movement from the first substrate is slow, and they become trapped on the first substrate, suppressing their movement to the second substrate. This allows for reliable fluorescence color development on the second substrate even with simple operations such as directly dropping blood onto the first substrate, enabling stable evaluation of the reaction.

[0033] The substrates used for the first and second substrates can be materials that allow the liquid of the test subject to flow and can appropriately support reagents, etc. The substrates used for the first and second substrates may be of the same type or different types. Various porous materials can be used as substrates. The shape of the substrate material can be a plate-like material, a thin film, a film, a sheet, etc. For example, porous membranes, woven fabrics, nonwoven fabrics, etc., can be used as substrates. As for the material of the substrate, a substrate that does not easily produce autofluorescence is preferred, and nitrocellulose membranes and glass fiber nonwoven fabrics can be used. There is no particular upper or lower limit to the thickness of the substrate, but thin films are fine considering the physical properties required for each substrate, for example, those of about 20 μm to 2000 μm or about 50 μm to 1000 μm can be used.

[0034] The first substrate and the second substrate in the detection member may be formed by bonding parts of separately manufactured materials together, as shown in Figure 2, or by processing one substrate so that a portion functions as the first substrate and the other portion functions as the second substrate. When loading reagents or other materials for the second substrate, the components of the substrate may be dispersed, so it is preferable to prepare the second substrate in advance, dry it, and then bond it to the substrate that will become the first substrate.

[0035] [Substrate for detecting quenched thrombin activity] A substrate for detecting quenched thrombin activity has a fluorescent group using a fluorescent reagent and a quenching group using a quenching reagent. Figure 5 shows an example of the assumed mechanism of action of the substrate for detecting quenched thrombin activity, which is Förster resonance energy transfer (FRET), and a structural example to explain the principle of the substrate for detecting quenched thrombin activity. In this structural example in Figure 5, under normal conditions, it is presumed that the fluorescent reagent structure Nma (N-methylacridone), chemically bonded to one end, does not emit light due to Förster resonance energy transfer (FRET) to the quenching reagent structure Dnp (2,4-dinitrophenyl) at the other end within the same molecule. However, it is presumed that if a partial separation of the structure occurs and the bond between the fluorescent reagent structure and the quenching reagent structure is broken, FRET will not occur, and the fluorescence emission of Nma will be restored. Therefore, the intensity of this emitted fluorescence is positively correlated with the amount of thrombin activity. Thus, a substrate for detecting quenched thrombin activity is a substrate that has the structure of a fluorescent reagent component and the structure of a quenching reagent component, and by introducing a structure that reacts with thrombin as a separation in that structure, fluorescence emission is produced when it reacts with thrombin.

[0036] When thrombin production is promoted by the second substrate, fluorescence emission occurs. On the other hand, if thrombin production is not activated, no fluorescence emission occurs. Therefore, since fluorescence is generated according to the degree of thrombin production by the subject in the second substrate, blood coagulation can be evaluated by assessing the degree of fluorescence coloration.

[0037] A substrate for detecting quenched thrombin activity is a substrate that is decomposed and separated by the generated thrombin into a fragment containing a fluorescent reagent and a fragment containing a quenching reagent. The luminescence of the fluorescent reagent is restored as the resonance energy transfer disappears due to the decomposition and separation of the substrate.

[0038] The substrate for detecting quenched thrombin activity can be an oligopeptide derivative. An oligopeptide derivative is formed by chemically bonding a fluorescent reagent and a quenching reagent to the amino acids at each terminal end of an oligopeptide, for example, by crosslinking.

[0039] [Oligopeptide derivatives] The oligopeptide in the oligopeptide derivative is preferably an oligopeptide with an amino acid sequence of about 5 to 20 units or a sequence length of about 8 to 15 units. It is also preferable that one terminal end has lysine or cysteine, and the other terminal end also has lysine or cysteine. Lysine or cysteine ​​is suitable as a target for chemical bonding of fluorescent reagents or quenching reagents. The amino acid to which the fluorescent reagent or quenching reagent is chemically bonded at this terminal end is preferably lysine. It is also preferable that there are lysine or cysteine ​​at both ends, separated by multiple amino acid residues. The lysine or cysteine ​​may be at the end of the amino acid sequence of the oligopeptide, or it may be located 1 to 3 units from the end.

[0040] Furthermore, it is preferable that the amino acid sequence includes a "-proline-arginine-" sequence. This sequence functions as a starting point for separation and decomposition into a fragment on the fluorescent reagent side and a fragment on the quenching reagent side when the testing material is used. It is also preferable that each end has a "-proline-arginine-" sequence via an arbitrary amino acid sequence of about 1 to 8 or 2 to 5. Thrombin tends to selectively cleave the "-proline-arginine-" amino acid sequence. Thrombin is directed towards proline, and arginine becomes the cleavage site.

[0041] When the substrate used to detect thrombin is a low molecular weight substrate consisting of 2 or 3 amino acids, it may not be suitable as a substrate for highly sensitive detection of thrombin activity on the substrate. This is thought to be because the adsorption capacity to the substrate used in the testing method is low, making it difficult to solidify a sufficient amount of substrate, and because the fluorescent substance released by the action of thrombin rapidly moves from the solid phase to the liquid phase. Using oligopeptides with amino acid sequences of about 5-20 or 8-15 amino acids makes them more stable when supported on the substrate, and even if part of the sequence separates or decomposes while in the liquid phase during use, the supported state is maintained, allowing for stable color development and detection.

[0042] [Amino acid sequence of oligopeptides] The amino acid sequence of an oligopeptide can be represented by the following basic sequences (1) and (2). [Basic Sequence (1)] (N-terminus) "Lysine" - "X1" - "Proline" - "Arginine" - "X2" - "Arginine" - "X3" - "Lysine" (C-terminus) [Basic Sequence (2)] (N-terminus) "Lysine" - "X1" - "Proline" - "Arginine" - "X4" - "Lysine" (C-terminus) In the above amino acid sequence, X1, X2, X3, and X4 are sometimes collectively referred to as X. X(X1, X2, X3, X4) can be any amino acid. X1-X4 can consist of approximately 1-5 residues, 1-3 residues, and 1-2 residues of amino acids, respectively.

[0043] The amino acids constituting X are preferably other amino acids, excluding lysine, cysteine, proline, and arginine. Specifically, the amino acids constituting X can be selected from the group consisting of alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

[0044] As a dried form of oligopeptide derivative, for example, one can be used in which fluorescent reagents or quenching reagents are crosslinked at each end of the following amino acid sequence (N-terminus → C-terminus), as described later.

[0045] [Example of peptide sequence (1)] "Lysine (crosslinked with fluorescent reagent N-methylacridone)" - "Leucine" - "Valine" - "Proline" - "Arginine" - "Serine" - "Tryptophan" - "Arginine" - "Leucine" - "Lysine (crosslinked with quenching reagent 2,4-dinitrophenyl)" (See Sequence ID (1))

[0046] [Example of peptide sequence (2)] "Lysine (crosslinked with fluorescent reagent N-methylacridone)" - "Leucine" - "Valine" - "Proline" - "Arginine" - "Serine" - "Phenylalanine" - "Arginine" - "Leucine" - "Lysine (crosslinked with quenching reagent 2,4-dinitrophenyl)" (See Sequence ID (2))

[0047] [Example of peptide sequence (3)] "Lysine (crosslinked with fluorescent reagent N-methylacridone)" - "Leucine" - "Tryptophan" - "Proline" - "Arginine" - "Glycine" - "Tryptophan" - "Lysine (crosslinked with quenching reagent 2,4-dinitrophenyl)" (See Sequence ID (3))

[0048] [Fluorescent reagents] Fluorescent reagents are reagents that react with thrombin and fluoresce when irradiated with excitation light. For example, N-methylacridone, fluorescein (FAM), Texas Red®-X, and Pulsar 650 can be used. Fluorescent reagents can be chemically bonded to oligopeptides to form fluorescent groups through chemical reactions such as condensation reactions.

[0049] [Quenching agent] Quenching reagents are reagents that bind to fluorescent reagents in substrates used for detecting quenched thrombin activity, inhibiting the reaction of the fluorescent reagents and thus quenching them. For example, 2,4-dinitrophenyl, Black Hole Quencher (BHQ®)-1, and Black Hole Quencher (BHQ®)-2 can be used. Quenching reagents can be chemically bonded to oligopeptides by chemical reactions such as condensation reactions.

[0050] Combinations of fluorescent groups and quenching groups can include, for example, "fluorescent group N-methylacridone-quenching group 2,4-dinitrophenyl," "fluorescent group Fluorescein-quenching group Black Hole Quencher-1," "fluorescent group Texas Red-X-quenching group Black Hole Quencher-2," and "fluorescent group Pulsar650-quenching group Black Hole Quencher-2."

[0051] Substrates for detecting quenchable thrombin activity, such as dried oligopeptide derivatives, are placed on a support substrate with a planar view of an area of ​​1 cm². 2 The wavelength range can be approximately 0.05 nmoles to 100 nmoles, 0.10 nmoles to 60.0 nmoles, or 0.11 nmoles to 57.2 nmoles.

[0052] [Coagulation activator] Coagulation activators are substances that activate blood coagulation by reacting with blood. These can include tissue factor (TF), which induces thrombin production, activated blood coagulation factors, and snake venom-derived activators. These may be used individually or in combination.

[0053] Activated blood coagulation factors can include, for example, activated blood coagulation factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, prekallikrein, protein C, and other enzymes and coenzymes involved in coagulation reactions. In particular, activated blood coagulation factor XI (FXIa) can be used.

[0054] Snake venom-derived activators can include, for example, coagulation activators derived from toxins used in diluted Russell's viper venom time tests and carpet viper venom-derived ecarin coagulation time tests. Russell's viper venom activates coagulation through the activation of coagulation factor X. Ecarin induces thrombin production by activating prothrombin.

[0055] Coagulation activating substances such as tissue factor are present in a planar view of the substrate surface area of ​​1 cm². 2 The values ​​can range from 0.05 fmoles to 200 fmoles, 0.10 fmoles to 100 fmoles, or 0.25 fmoles to 62.8 fmoles.

[0056] The temperature during the thrombin production test using the test components can be set to approximately 20-45°C, preferably 23-40°C, more preferably 35-40°C, and particularly preferably 37-38°C, at which point the reactions for thrombin production and thrombin activity expression occur. The reaction time can be approximately 1-30 minutes, and can be 1-10 minutes or 5-10 minutes.

[0057] The testing component of the present invention may contain other components. The testing component is typically used in tests that react in a water-based environment. Therefore, the medium of the testing component may be primarily water. It may also contain adsorption inhibitors and protective agents for proteins such as FXIa and TF, pH adjusters, mineral adjusters, etc. For example, it may contain serum albumin, buffer solutions, synthetic phospholipids, calcium chloride, etc.

[0058] [Inspection equipment (20)] The testing device contains components for blood coagulation testing and is positioned in a predetermined location. By using the testing device, a sample can be supplied and evaluated more easily and reliably. The testing device only needs to accommodate the first and second substrate sides of the testing components in alignment with the supply and observation sections, and can be a box-shaped container or the like that houses thin, plate-like components with all openings except those corresponding to the supply and observation sections closed.

[0059] [Supply Department (21)] The supply unit is the part for supplying the sample to the first substrate. The supply unit can be, for example, an opening that serves as a flow path located at a position corresponding to the first substrate. By supplying the sample from the supply unit, the sample comes into contact with the first substrate.

[0060] [Observation Section (22)] The observation section is for observing the reaction state of the second substrate. Since the observation section is for observing the fluorescence produced when excitation light is irradiated onto it, it can have an opening large enough to allow sufficient visual inspection of the second substrate. The observation section can have any shape, such as circular or polygonal. Excitation light from a fluorescent reagent is irradiated onto the observation section, and the fluorescence emission state of the second substrate is detected from the observation section.

[0061] [Blood coagulation assessment] The determination of blood coagulation is a process that evaluates the state of blood coagulation by comparing it with an index that assesses the state of blood coagulation based on the luminescence state of a second substrate, such as an observation unit. Fluorescence can also be observed visually under certain conditions. The index may simply be the presence or absence of fluorescence. Alternatively, the amount of thrombin produced, which is involved in blood coagulation, can be determined by comparing it with an intensity table that determines the degree of fluorescence.

[0062] [Inspection Flow] Figure 4 is a schematic diagram of the inspection method according to the present invention. The inspection method of the present invention can be performed using the inspection member of the present invention. Step S11 is the process of supplying the subject to the first substrate. Step S21 is the process of irradiating the second substrate with excitation light. Step S31 is the process of detecting the luminescence state of the second substrate. Step S41 is the process of evaluating the state of blood coagulation based on the luminescence state.

[0063] When a sample is brought into contact with the first substrate, some of the sample's components move from the first substrate to the second substrate. These components then react with the reagent on the second substrate. The second substrate is then irradiated with excitation light, and the fluorescence state is observed. From this fluorescence state, the blood coagulation components of the sample can be evaluated.

[0064] The blood coagulation test according to the present invention can be performed to support the prevention and treatment of congenital or acquired hemorrhagic diseases (hemorrhagic bleeding). For example, by performing this test in the treatment of thrombosis (such as cerebral infarction and myocardial infarction) using anticoagulant drugs, the risk of bleeding due to medication can be evaluated and determined, and the type and amount of medication can be optimized for each patient, thereby achieving safe treatment that prevents excessive bleeding.

[0065] Furthermore, it can be used for screening patients with congenital bleeding disorders, such as hemophilia, and for evaluating the effectiveness of therapeutic drugs for these patients.

[0066] Furthermore, this test is also very useful in determining the risk of bleeding caused by DOACs. The thrombin formation test is a blood coagulation activity measurement method that quantifies the thrombin formed by adding a calcium-containing thrombin-forming reagent to the blood or plasma of the test subject and allowing it to react for a certain period of time.

[0067] This test measures the activity level of the prothrombinase complex in the blood, as the prothrombinase complex generates thrombin. While various reagents and methods for thrombin formation tests have been investigated, no test has been developed that can evaluate and determine the bleeding risk caused by DOACs.

[0068] The inventors have invented a groundbreaking thrombin formation test with new performance by improving the test reagent used in the thrombin formation test. We believe this thrombin formation test will contribute to the realization of safer, more personalized treatment with reduced side effects in DOAC therapy.

[0069] This invention relates to a point-of-care coagulation test that can be performed without requiring complex or advanced procedures in small medical facilities such as hospitals or clinics, at the bedside, in homes, and in emergency medical settings, and to obtain test results quickly, thereby enabling earlier and more appropriate treatment strategy decisions. [Examples]

[0070] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless its essence is changed.

[0071] Reference Example 1: Fluorescent substrates used for detecting thrombin activity in thrombin production tests, such as "glycyl-glycyl-arginyl-aminomethylcoumarin and D-cyclohexylalanyl-alanyl-arginyl-aminomethylcoumarin," have a structure in which the fluorescent substance aminomethylcoumarin is bonded to the carboxyl terminus of arginine.

[0072] Thrombin cleaves and decomposes these substrates between arginine and aminomethylcoumarin, releasing aminomethylcoumarin from the substrate and causing fluorescence emission. While these substrates are very useful in thrombin production studies under liquid phase conditions such as blood, their low molecular weight makes it difficult to directly immobilize them on supports such as membranes.

[0073] Example 1: Oligopeptide substrate for detecting quenchable thrombin activity used in this test Based on the following experiments, the inventors have discovered an evaluation method using a high molecular weight oligopeptide substrate that has higher thrombin specificity and can be more stably immobilized on a support without chemical bonding.

[0074] To prevent the fluorescent substance released from the substrate from rapidly migrating from the reagent pad support to the liquid phase of blood, a quenchable thrombin activity detection substrate was invented. This substrate is an oligopeptide derivative that, upon the action of thrombin, is decomposed and separated into a fragment containing a fluorescent reagent and a fragment containing a quenching reagent, and the luminescence of the fluorescent reagent is restored by the disappearance of Förster resonance energy transfer (FRET) (Non-patent literature: Anal. Biochem., 50:56-62, 1972; see Figure 2).

[0075] [Test Method] (1) Preparation and characterization of oligopeptide substrates for detection of quenchable thrombin activity 1) Preparation of oligopeptide substrates In the present invention, the following oligopeptide substrates for detecting quenchable thrombin activity were prepared. These substrates were prepared by synthesizing oligopeptides using a peptide synthesizer (ABI433A, Applied Biosystems), and then crosslinking the amino groups of the side chains of lysine residues in the peptides with a fluorescent reagent and a quenching reagent, respectively, by condensation reactions.

[0076] The fluorescent reagent N-methylacridone (Nma) was introduced into the amino-terminal lysine residue, while the quenching reagent 2,4-dinitrophenyl (Dnp) was introduced into the carboxy-terminal lysine residue. The carboxyl group in the carboxy-terminal lysine residue was modified with an amide group. The oligopeptide substrate was purified by high-performance liquid chromatography (LC-8A, Shimadzu Corporation) using a reversed-phase column (YMC-Pack ODS-A 30X250mm, YMC). 0.1% trifluoroacetic acid (TFA) and 0.1% TFA containing 70% acetonitrile were used as solvents.

[0077] • Substrate #1: Lysine (Nma)-Leucine-Valine-Proline-Arginine-Serine-Tryptophan-Arginine-Leucine-Lysine (Dnp)-Amide group • Substrate #2: Lysine (Nma)-Leucine-Valine-Proline-Arginine-Serine-Phenylalanine-Arginine-Leucine-Lysine (Dnp)-Amide group • Substrate #3: Lysine (Nma)-Leucine-Tryptophan-Proline-Arginine-Glycine-Tryptophan-Lysine (Dnp)-Amide group

[0078] 2) Analysis of the physicochemical properties of oligopeptide substrates for detecting quenchable thrombin activity The purity of the oligopeptide substrate was analyzed by high-performance liquid chromatography (LC-20A, Shimadzu Corporation) using a reversed-phase column (Zorbax300SB-C18: 4.6 mm × 150 mm, Agilent). The molecular weight was determined by electrospray ionization mass spectrometry (ESI-MS: InfinityLabLC / MSDG6125B, Agilent Technologies, Inc.). The amino acid composition of the oligopeptide substrate was analyzed by hydrolyzing the oligopeptide substrate with 4N methanesulfonic acid at 150°C for 2.5 hours, followed by analysis using an amino acid composition analyzer (High-Speed ​​Amino Acid Analyzer L-8900, Hitachi, Ltd.).

[0079] 3) Analysis of the reactivity of oligopeptide substrates for detecting quenchable thrombin activity with thrombin The reactivity of thrombin (Prolytix) with the oligopeptide substrate for detecting quenchable thrombin activity was investigated using the following method. The substrates to be tested were diluted with 50 mM Tris buffer (EDTA / TBS, pH 7.4) containing 27 mM EDTA and 0.15 M sodium chloride, while α-thrombin and other activated coagulation factors (activated coagulation factor IX, activated coagulation factor X, activated coagulation factor XI, activated protein C: purchased from Prolytix) were diluted with TBS (BSA / TBS, pH 7.4) containing 0.5% bovine serum albumin.

[0080] 3-1) Investigation of substrate concentration dependence in reaction with thrombin under liquid phase conditions 50 μL of substrate solutions #1 to #3 at various concentrations were dispensed into a 96-well microtiter plate and heated at 37°C for 2 minutes. After heating, 55 μL of α-thrombin (8,000 pM) was added to the substrates and the reaction was allowed to proceed at 37°C for 2 minutes. During the 2-minute reaction, the fluorescence intensity produced by the cleavage and decomposition of the substrate by α-thrombin was measured over time at 12-second intervals using a fluorescence plate reader (BioTek Synergy HTX reader, excitation wavelength: 360 nm, fluorescence wavelength: 460 nm). After the measurement was completed, the rate of change in fluorescence intensity per unit time (slope: fluorescence intensity / second) was calculated. The rate of change in fluorescence intensity is positively correlated with the activity level of thrombin.

[0081] 3-2) Specificity of thrombin for the substrate that has been solidified and dried on a support. The oligopeptide substrates for detecting quenchable thrombin activity were immobilized on a support such as a membrane, dried, and the reactivity of substrates #1 to #3 with thrombin was examined. Furthermore, the specificity of thrombin was investigated by comparing the reactivity with other activated coagulation factors. In fact, a cellulose membrane (28 mm) was used. 2 500 μL of substrate solution (100 μM or 200 μM) was added to a solution (either from Advantec or Cytiva) and incubated at 37°C for 1 hour. The substrate solution was diluted and prepared using TBS buffer. After incubation, the substrate solution was removed from the membrane, and the membrane was washed twice with 1 mL of phosphate buffer (PBS, pH 7.4). After washing, the membrane was dried at room temperature, and the reactivity of the solid-phase, dried substrates #1 to #3 with thrombin was examined. The reactivity with thrombin was assessed by adding a dried film containing the substrate to a 96-well microtiter plate, then adding 100 μL of α-thrombin (31 pM to 8,000 pM). The fluorescence intensity resulting from the cleavage and degradation of the substrate by α-thrombin was measured over 5 minutes at 15-second intervals using a fluorescence plate reader. The measurements were performed at 37°C. After the measurement was completed, the rate of change in fluorescence intensity per second (fluorescence intensity / second) was calculated.

[0082] The specificity of thrombin was investigated using substrate #1, which was solid-phase and dried on a glass fiber membrane. 2 100 μL of substrate solution (200 μM) was added to a cytiva (Cytiva) and incubated at 37°C for 1 hour. The substrate solution was diluted and prepared using TBS buffer. After incubation, the substrate solution was removed, and the membrane was dried at room temperature to prepare a glass fiber membrane containing solid-phase, dried substrate. To investigate the reactivity with thrombin, the above glass fiber membrane was added to a 96-well microtiter plate, and 40 μL of α-thrombin, activated coagulation factor IX, activated coagulation factor X, activated coagulation factor XI, or activated protein C (125 pM to 4,000 pM) was added. The fluorescence intensity produced by the action of these enzymes was measured over 5 minutes at 15-second intervals using a fluorescence plate reader. The measurements were performed at 37°C. After the measurement was completed, the rate of change in fluorescence intensity per second (fluorescence intensity / second) was calculated.

[0083] [Test Results] 1) Physicochemical properties of oligopeptide substrates for detecting quenchable thrombin activity The oligopeptide substrates for detecting quenchable thrombin activity prepared in this invention had the following physicochemical properties. Since these substrates had a high purity of over 98%, they were used to investigate their reactivity with activated coagulation factors such as thrombin.

[0084] • Substrate #1: Lysine (Nma)-Leucine-Valine-Proline-Arginine-Serine-Tryptophan-Arginine-Leucine-Lysine (Dnp)-Amide group Molecular weight: 1580.9, Purity: 99.6%, Amino acid composition analysis: Matches the composition of the synthesized peptide. • Substrate #2: Lysine (Nma)-Leucine-Valine-Proline-Arginine-Serine-Phenylalanine-Arginine-Leucine-Lysine (Dnp)-Amide group Molecular weight: 1541.9, Purity: 98.6%, Amino acid composition analysis: Matches the composition of the synthesized peptide. • Substrate #3: Lysine (Nma)-Leucine-Tryptophan-Proline-Arginine-Glycine-Tryptophan-Lysine (Dnp)-Amide group Molecular weight: 1368.5, Purity: 99.4%, Amino acid composition analysis: Matches the composition of the synthesized peptide.

[0085] 2) Analysis of the reactivity of oligopeptide substrates for detecting quenchable thrombin activity with thrombin

[0086] 2-1) Investigation of substrate concentration dependence in reaction with α-thrombin under liquid phase conditions Figure 6 shows the results of investigating the reactivity of α-thrombin (8,000 pM) with substrates #1 and #3 under liquid-phase conditions. It was found that for all substrates, increasing the substrate concentration increased the thrombin activity, expressed as the rate of change in fluorescence intensity (fluorescence intensity / second). Substrate #1 showed a higher rate of change in fluorescence intensity even at low concentrations of 20 μM or less compared to substrate #3, suggesting it has a high affinity for thrombin.

[0087] Figure 6 is a graph showing the substrate concentration dependence of α-thrombin under liquid-phase conditions. The reactivity of α-thrombin (8,000 pM) with substrates #1 and #3 under liquid-phase conditions was investigated. Each point represents the mean and standard deviation of thrombin activity expressed as fluorescence intensity / second (number of measurements = 3-4).

[0088] 2-2) Reactivity of α-thrombin with solid-phase, dried substrates on cellulose membranes The reactivity of α-thrombin with solid-phase, dried substrates #1 and #3 on a cellulose membrane was investigated. Figure 7 shows the time course of fluorescence intensity after adding α-thrombin (8,000 pM) to a cellulose membrane containing the dried substrates. Upon adding thrombin and initiating the reaction, the Nma fluorescence value increased in a time-dependent manner. In addition, the rate of increase in fluorescence intensity also increased with increasing solid-phase substrate concentration. This indicates that α-thrombin cleaved the arginine-serine bond in substrate #1 and the arginine-glycine bond in substrate #3, causing the Nma-containing fragment of the fluorescent reagent located at the amino-terminus of the substrate to separate from the Dnp fragment of the quenching reagent, thus restoring luminescence. Furthermore, the rate of increase in fluorescence intensity for substrate #1 was significantly higher than that for substrate #3. This indicates that thrombin can be detected with higher sensitivity in a test system where substrate #1 is solid-phase and dried on a cellulose membrane.

[0089] Figure 7 is a graph showing the reactivity of α-thrombin to substrates #1 and #3, which were solid-phase and dried on a cellulose membrane. Figure 7A shows the reactivity of 100 μM or 200 μM substrate #1 with α-thrombin over time after solid-phase drying on a cellulose film. Figure 7B shows the reactivity of α-thrombin to solid-phase, dried substrate #3 (100 μM or 200 μM). Each point shows the average and standard deviation values ​​of the fluorescence (number of measurements = 2).

[0090] 2-3) Concentration dependence and specificity of thrombin on the substrate solid-phase and dried on a glass fiber membrane The reactivity of α-thrombin, activated coagulation factor IX, activated coagulation factor X, activated coagulation factor XI, and activated protein C with substrate #1, which was solid-phase and dried on a glass fiber membrane, was investigated. When α-thrombin of various concentrations was added to the membrane and reacted, the rate of change in fluorescence intensity increased in a concentration-dependent manner with α-thrombin (Figure 8). Furthermore, since the rate of change increased linearly with concentration, it was revealed that the amount of thrombin could be quantified with high accuracy in the concentration range from 125 pM to 4,000 pM. On the other hand, activated coagulation factor IX, activated coagulation factor X, activated coagulation factor XI, and activated protein C hardly reacted with this substrate. It was found that substrate #1 has very high specificity for α-thrombin.

[0091] Figure 8 is a graph showing the concentration dependence and specificity of thrombin on solid-phase dried substrate #1 on a glass fiber membrane. The reactivity of α-thrombin, activated coagulation factor IX, activated coagulation factor X, activated coagulation factor XI, and activated protein C to solid-phase dried substrate #1 was investigated. Each point represents the mean and standard deviation (number of measurements = 3-6) of the hydrolysis activity (fluorescence intensity / second) of the substrate.

[0092] Example 2: Thrombin production test performed using a sensor in which a coagulation activating substance and an oligopeptide substrate for detecting quenchable thrombin activity were solid-phase and dried on a support such as a membrane.

[0093] Sensors (hereinafter referred to as "test components") were fabricated by solid-phase drying a coagulation activating substance and an oligopeptide substrate for detecting quenchable thrombin activity (substrate #1 or #2 mentioned above) on a glass fiber membrane support, and thrombin production tests were conducted using plasma. Two types of test components were fabricated in this study.

[0094] • Component (1) Test component consisting only of a second substrate containing a coagulation activating substance and a thrombin activity detection substrate. • Component (2) is a testing component consisting of a first substrate for dropping and contacting a plasma sample, and a second substrate containing a coagulation activating substance and a substrate for detecting thrombin activity.

[0095] The amount of thrombin produced in plasma was quantified by measuring the intensity of fluorescence emitted when the detection substrate is cleaved and decomposed by the action of thrombin, using a fluorescence plate reader. In this invention, tissue factor (TF) assay reagent (Dade Innovin: Sysmex Corporation) was used as the coagulation activator.

[0096] [Test Method] (1) Preparation of a second test substrate consisting of a coagulation activator and an oligopeptide substrate for detecting thrombin activity The second substrate for testing contains a solid-phase, dried coagulation activator and an oligopeptide substrate for detecting quenchable thrombin activity. The second substrate acts as a reagent pad support in the measurement area. In fact, the second substrate is a glass fiber membrane (2.8 cm 2 A 100 μL mixed solution containing tissue factor (TF) test reagents (110 pM, 440 pM) and oligopeptide substrate #1 or #2 (100 μM, 200 μM) for detecting quenchable thrombin activity was added to a cytiva film and incubated at 37°C for 1 hour. After that, the mixed solution was removed and the film was dried at room temperature to prepare a solid-phase, dried second substrate for testing. The mixed solution was prepared by mixing the TF test reagent solution with the oligopeptide substrate solution in a 1:4 ratio. The TF test reagent solution and the oligopeptide substrate solution were also prepared by diluting them with TBS buffer (pH 7.4).

[0097] (2) Preparation of a test component consisting of a first base material for dropping blood samples and a second base material containing reaction reagents. Figure 2 shows a schematic diagram of a testing component consisting of a first substrate for dropping plasma samples and a second substrate containing reaction reagents. This testing component has a first substrate (glass fiber membrane, 3.2 cm) for dropping blood (plasma) samples. 2The first substrate (manufactured by Cytiva) was placed on the second substrate prepared using the method described above, overlapping it by 0.5 mm to 5 mm, and the two films were brought into direct contact. This contact causes the blood (plasma) sample dropped onto the first substrate to move to the second substrate by capillary action and react with reagents supported on the second substrate. The second substrate consists of a dried fluorescence resonance energy transfer type quenchable thrombin activity detection substrate and a coagulation activator, which are solid-phase and dried, and when integrated with the first substrate, it becomes a luminescent biosensor.

[0098] (3) Thrombin production test of plasma using testing components 1) Analysis of thrombin production in plasma using a test component consisting only of the second substrate. To perform a plasma thrombin production test using the test material of the second substrate, first, the dried second substrate was added to the wells of a 96-well microtiter plate. Next, 70 μL of normal control plasma (Simens blood coagulation test control plasma N: containing the anticoagulant citrate) and 20 μL of BSA / TBS buffer were added to the plate wells and heated at 37°C for 2 minutes. After heating, 20 μL of calcium chloride solution (88 mM) was added to investigate thrombin production in the second substrate, and the mixture was reacted at 37°C for 10 minutes. During this time, thrombin was produced in the plasma, and the produced thrombin cleaved and degraded the oligopeptide substrate for detecting thrombin activity, causing fluorescence. The intensity of the resulting fluorescence was measured over time at 15-second intervals using a fluorescence plate reader (BioTek Synergy HTX reader, excitation wavelength: 360 nm, fluorescence wavelength: 460 nm). The measurements were performed at 37°C. After the measurement was completed, the rate of change in fluorescence intensity per unit time (fluorescence intensity / second) was calculated. The rate of change in fluorescence intensity is positively correlated with the amount of thrombin produced in plasma.

[0099] 2) Analysis of thrombin production in plasma using a test component composed of a first substrate and a second substrate. To test thrombin production in plasma within the test material, 40 μL of plasma (with 88 mM calcium chloride added) was first dropped onto the first substrate and allowed to stand at room temperature for 3 minutes. During this time, the plasma migrated to the second substrate and reacted with the reagents in the second substrate. After 3 minutes, the second substrate was removed from the test material and transferred to a 96-well microtiter plate. The fluorescence intensity produced by the action of thrombin was then measured over 5 minutes at 15-second intervals using a fluorescence plate reader, in the same manner as described above. The measurements were performed at room temperature (~25°C).

[0100] [Test Results] (1) Thrombin production of plasma using the testing component of the second substrate Figure 9 shows the results of thrombin production in a second test substrate prepared using a solution of oligopeptide substrates for detecting quenchable thrombin activity (200 μM each) mixed with TF test reagent (110 pM or 440 pM). In the second substrate containing substrate #1, thrombin production increased in a time-dependent manner, and this increase also depended on the concentration of the TF reagent in the mixture (Figure 9(a)). TF-dependent thrombin production was significantly reduced by the addition of an anti-TF monoclonal antibody (10 μg / mL) that inhibits TF activity, and this was supported by the results of the rate of change in fluorescence (fluorescence intensity / second) calculated between 100 and 240 seconds (Figure 9(c)). Thrombin production in the second substrate containing substrate #2 increased in a time-dependent manner, similar to substrate #1, and this increase also depended on the concentration of the TF reagent in the mixture (Figure 9(b), (c)). However, the suppression of thrombin production by the addition of the anti-TF antibody was not as pronounced as with substrate #1. In the case of substrate #2, the onset time of thrombin production was earlier, which was thought to be due to the low efficiency of antibody inhibition. The second substrate for this test, using substrate #1, was usable for the analysis of thrombin production in plasma.

[0101] Figure 9 is a graph showing the thrombin production test of plasma using a test component of the second substrate containing the reaction reagent. (a) Plasma was added to the second substrate of substrate #1, and the Nma fluorescence value was measured over time. The measured values ​​are average values ​​(number of measurements = 2). (b) Thrombin production test on the second substrate of matrix #2. The measurement is the same as (a). (c) From the results of (a) and (b), the change rate of fluorescence intensity (fluorescence intensity / second) was calculated to quantify the amount of thrombin produced. The anti-TF monoclonal antibody significantly inhibited thrombin production.

[0102] (2) Thrombin production in plasma using a test member composed of a first substrate and a second substrate The thrombin production in plasma was analyzed using a test member prepared by combining the first substrate and the second substrate. For the purpose of optimizing the amount of TF reagent and substrate reagent immobilized on the second substrate, 2.5 fmoles / cm 2 ~157 fmoles / cm 2 of TF reagent and 1.1 nmoles / cm 2 ~143 nmoles / cm 2 of matrix #1 were used. When plasma was dropped onto the first substrate, the amount of thrombin produced increased time-dependently in the second substrate, and this increase was completely inhibited by the addition of anti-TF antibody (Figure 10(a)). Furthermore, an increase in the amount of thrombin produced depending on the amount of TF immobilized (Figure 10(b)) and the amount of matrix #1 (Figure 10(c)) was confirmed. The test member composed of the first substrate and the second substrate was very useful in the POCT-type thrombin production test.

[0103] Figure 10 is a graph showing the thrombin production test in plasma using a test member composed of a first substrate and a second substrate. A test member was prepared by combining the first substrate onto which plasma was dropped and the second substrate containing the reaction reagent, and the thrombin production in plasma was analyzed using it. On the second substrate, 2.5 fmoles / cm 2 ~157 fmoles / cm 2 of TF reagent and 1.1 nmoles / cm 2 ~143 nmoles / cm 2 of matrix #1 were immobilized. (a) Time-dependence of the amount of thrombin produced. Each point shows the average value of fluorescence values (number of measurements = 2 - 4). (b) Dependence of thrombin production on the amount of immobilized TF. Results are shown as mean and standard deviation (number of measurements = 2-4). (c) Dependence of thrombin production on the amount of immobilized substrate #1. Results are shown as mean and standard deviation (number of measurements = 2).

[0104] (3) Evaluation of the anticoagulant effect of new generation antithrombotic drugs using this test component A point-of-care (POCT) coagulation test method for evaluating the anticoagulant effects of DOACs, which are widely prescribed as next-generation antithrombotic drugs, has yet to be established. In this invention, we demonstrated that a thrombin production test using a POCT testing component can test the anticoagulant effects of DOACs (rivaroxaban and apixaban) with high sensitivity. In fact, when plasma containing DOACs was dropped onto the first substrate, the amount of thrombin produced in the second substrate was significantly lower compared to the control without DOACs, and this decrease was DOAC concentration-dependent (Figure 11(a), (b)). Furthermore, it became clear that this invention is a highly sensitive coagulation test that can evaluate the anticoagulant effects of extremely low concentrations of DOACs that have not been detected before.

[0105] Figure 11 is a graph showing the evaluation of the anticoagulant effect of new-generation antithrombotic drugs (DOACs). The anticoagulant effect of DOACs (rivaroxaban and apixaban) was evaluated using a POCT (point-of-care testing) component consisting of a first base material and a second base material. (a) Effects of DOACs on thrombin production. Plasma containing rivaroxaban (3.1 ng / mL) or apixaban (12.5 ng / mL) was dropped onto the first substrate, and the fluorescence values ​​emitted from the second substrate were measured over time. The mean and standard deviation (number of measurements = 2) are shown. (b) DOACs suppressed thrombin production (rate of change) in a concentration-dependent manner. The columns show the mean and standard deviation (number of measurements = 2). Significant difference tests between two or more groups were performed using one-way ANOVA, and a p-value < 0.05 was considered statistically significant. [Industrial applicability]

[0106] The testing components of the present invention can be used for blood coagulation testing and have industrial applicability. Furthermore, the testing method of the present invention is a method of collecting various data by analyzing samples taken from humans, and does not constitute a medical act performed by a physician on a human being, nor does it fall under the category of surgery, treatment, or diagnosis of a human being, and therefore has industrial applicability. [Explanation of Symbols]

[0107] 10 Inspection components 11 First substrate 12 Second base material 13 Contact area 20 Inspection Instruments 21 Supply section 22 Observation Section

Claims

1. A first substrate for contacting a subject containing blood components, A blood coagulation testing component comprising a second substrate that contacts the first substrate and contains a quenchable thrombin activity detection substrate and a coagulation activating substance.

2. The aforementioned substrate for detecting quenching thrombin activity is The inspection member according to claim 1, which is an oligopeptide derivative that, upon contact with thrombin, separates into a fragment containing a fluorescent reagent and a fragment containing a quenching reagent, thereby causing the fluorescent reagent to emit light.

3. The inspection member according to claim 2, wherein the coagulation activating substance is one or more selected from the group consisting of tissue factor (TF), activated blood coagulation factor, and snake venom-derived activating substance, which react with blood to induce thrombin production.

4. A blood coagulation test component according to any one of claims 1 to 3 is arranged, A supply unit for supplying the subject to the first substrate, A blood coagulation testing device having an observation section for observing the reaction state of the second substrate.

5. A method for testing blood coagulation using a testing component comprising a first substrate for contacting a subject containing blood components, and a second substrate that contacts the first substrate and contains a quenchable thrombin activity detection substrate and a coagulation activating substance, The process of supplying a subject to the first substrate, The second substrate is subjected to the step of irradiating it with excitation light, A step of detecting the light emission state of the second substrate, A method for testing blood coagulation, comprising the steps of determining blood coagulation by comparing the luminescence state with an index that evaluates the state of blood coagulation.

Images