Reagent for coagulation time measurement and coagulation time measurement method

Abstract

This reagent for coagulation time measurement contains ellagic acid and at least one of a polymer and an amino acid lacking an aromatic ring. The content of the amino acid contained in the reagent for coagulation time measurement is preferably 20,000-270,000 parts by mass with respect to 100 parts by mass of the ellagic acid content. The content of the polymer contained in the reagent for coagulation time measurement is preferably 10-1,000 parts by mass with respect to 100 parts by mass of the ellagic acid content, and the reagent for coagulation time measurement does not contain phenol.

Description

Reagent for Measuring Coagulation Time and Method for Measuring Coagulation Time

[0001] The present invention relates to a reagent for measuring coagulation time and a method for measuring coagulation time. This application claims priority from Japanese Patent Application No. 2024-191854 filed in Japan on October 31, 2024, and incorporates its contents herein by reference.

[0002] Blood coagulation tests are tests for diagnosing a patient's blood coagulation ability by adding a predetermined reagent to a patient's blood sample and measuring the blood coagulation time and the like. Typical examples of blood coagulation time include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and the like. Abnormalities in blood coagulation ability cause an extension of the coagulation time. Causes of an extension of the coagulation time include coagulation inhibitors (e.g., heparin, etc.), a decrease in coagulation-related components, congenital deficiency of blood coagulation factors (e.g., hemophilia, etc.), autoantibodies that inhibit the coagulation reaction (e.g., lupus anticoagulant; LA, anticoagulation factor antibodies, etc.), and the like.

[0003] In blood coagulation tests, a reagent is added to a blood sample, and then the subsequent blood coagulation reaction is measured to measure the blood coagulation time. When an extension of the coagulation time is observed, it is determined that there is an abnormality in the blood coagulation ability. Blood coagulation test reagents contain components for advancing blood coagulation under certain conditions. For example, APTT measurement reagents contain coagulation factor activators such as ellagic acid, phospholipids, and buffers. On the other hand, there are various types of commercially available APTT measurement reagents, and the sensitivities of these reagents to various coagulation abnormalities are different.

[0004] Ellagic acid, which is a coagulation factor activator, is very difficult to dissolve in water or common organic solvents. Therefore, in reagents containing ellagic acid, there is a problem that precipitation of ellagic acid easily occurs. When precipitation of ellagic acid occurs, the ellagic acid used in the coagulation reaction is insufficient, so the progress of the coagulation reaction becomes slow, and as a result, the coagulation time is extended.

[0005] Patent Document 1 discloses solubilizing ellagic acid using phenol.

[0006] Patent document 2 discloses that ellagic acid can be solubilized by using an amino acid having an aromatic ring instead of phenol.

[0007] Japanese Patent Publication No. 5-506309, Japanese Patent Publication No. 2013-205087

[0008] Patent Document 1 describes the solubilization of ellagic acid using phenol. However, phenol is an environmentally harmful substance, and regulations on its use are becoming stricter.

[0009] Patent Document 2 describes solubilizing ellagic acid using an amino acid having an aromatic ring instead of phenol. However, the amino acid used is limited to those having an aromatic ring, and there is no disclosure regarding solubilizing ellagic acid using an amino acid without an aromatic ring.

[0010] The object of the present invention is to provide a novel reagent for measuring coagulation time and a method for measuring coagulation time that can solubilize ellagic acid without containing phenol.

[0011] The present invention encompasses the following embodiments: [1] A reagent for measuring coagulation time, comprising ellagic acid and at least one of an amino acid and a polymer that does not have an aromatic ring, and not containing phenol. [2] The reagent for measuring coagulation time according to [1], comprising the amino acid, wherein the content of the amino acid is 20,000 to 270,000 parts by mass per 100 parts by mass of the ellagic acid. [3] The reagent for measuring coagulation time according to [1] or [2], comprising the polymer, wherein the content of the polymer is 10 to 1,000 parts by mass per 100 parts by mass of the ellagic acid. [4] The reagent for measuring coagulation time according to [1] to [3], comprising the amino acid, wherein the amino acid is at least one amino acid selected from the group consisting of glycine, proline, threonine, valine, and methionine. [5] The coagulation time measuring reagent according to [1] to [4], wherein the polymer is at least one polymer selected from the group consisting of bovine serum albumin, Blocking Peptide Fragment, and chemically synthesized polymers. [6] The coagulation time measuring reagent according to [1] to [5], further comprising a metal salt compound. [7] The coagulation time measuring reagent according to [6], wherein the metal salt compound is at least one metal salt compound selected from the group consisting of manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate, and hydrates thereof. [8] A method for measuring coagulation time, comprising the steps of: obtaining a sample solution by mixing a blood sample with a coagulation time measuring reagent; and measuring the coagulation time of the sample solution obtained by the above step, wherein the coagulation time measuring reagent comprises ellagic acid and at least one of an amino acid and a polymer that does not have an aromatic ring, and does not contain phenol. [9] The method for measuring coagulation time according to [8], wherein the reagent for measuring coagulation time comprises a first reagent and a second reagent, the first reagent comprises ellagic acid and at least one of an amino acid and a polymer that does not have an aromatic ring, and does not contain phenol, and the second reagent comprises a calcium salt.

[10] The method for measuring coagulation time according to [9], wherein the step of obtaining the sample solution is to obtain a mixed solution by mixing the blood sample and the first reagent, and then mix the mixed solution with the second reagent.

[11] The method for measuring coagulation time according to any one of [8] to

[10] , wherein the reagent for measuring coagulation time contains the amino acid, and the content of the amino acid in the reagent for measuring coagulation time is 20,000 to 270,000 parts by mass per 100 parts by mass of the ellagic acid content.

[12] The method for measuring coagulation time according to any one of [8] to

[11] , wherein the reagent for measuring coagulation time contains the polymer, and the content of the polymer in the reagent for measuring coagulation time is 10 to 1,000 parts by mass per 100 parts by mass of the ellagic acid content.

[13] The method for measuring coagulation time according to any one of [8] to

[12] , wherein the reagent for measuring coagulation time comprises the amino acid, and the amino acid is at least one amino acid selected from the group consisting of glycine, proline, threonine, valine, and methionine.

[14] The method for measuring coagulation time according to any one of [8] to

[13] , wherein the reagent for measuring coagulation time comprises the polymer, and the polymer is at least one polymer selected from the group consisting of bovine serum albumin, Blocking Peptide Fragment, and chemically synthesized polymers.

[15] The method for measuring coagulation time according to any one of [8] to

[14] , wherein the reagent for measuring coagulation time further comprises a metal salt compound.

[16] The method for measuring coagulation time according to

[15] , wherein the metal salt compound is at least one metal salt compound selected from the group consisting of manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate, and hydrates thereof.

[17] A reagent for measuring coagulation time according to any one of [1] to [7], comprising the amino acid, wherein the content of the amino acid is 40,000 to 170,000 parts by mass per 100 parts by mass of the ellagic acid.

[18] A reagent for measuring coagulation time according to any one of [1] to [7] and

[17] , comprising the polymer, wherein the content of the polymer is 300 to 700 parts by mass per 100 parts by mass of the ellagic acid.

[19] The method for measuring coagulation time according to any one of [8] to

[16] , wherein the reagent for measuring coagulation time contains the amino acid, and the content of the amino acid is 40,000 to 170,000 parts by mass per 100 parts by mass of the ellagic acid content in the reagent for measuring coagulation time.

[20] The method for measuring coagulation time according to any one of [8] to

[16] and

[19] , wherein the reagent for measuring coagulation time contains the polymer, and the content of the polymer is 300 to 700 parts by mass per 100 parts by mass of the ellagic acid content in the reagent for measuring coagulation time.

[0012] According to the above embodiment, a novel reagent for measuring coagulation time and a method for measuring coagulation time can be provided, which can solubilize ellagic acid even without containing phenol.

[0013] In the following text, "amino acid without an aromatic ring" means an amino acid that does not have an aromatic ring in its side chain. Examples of such aromatic rings include benzene rings, fused benzene rings, non-benzene aromatic rings, and heteroaromatic rings.

[0014] In this specification, "polymer" refers to a molecule composed of multiple repeating units derived from monomers that are covalently bonded to one another. Furthermore, since "protein" is formed by the polymerization of amino acids, which are monomers, "protein" is a type of "polymer."

[0015] In the following specification, "blood coagulation time" may be simply referred to as "coagulation time." Similarly, "blood coagulation activator" may be simply referred to as "activator."

[0016] In the following specification, "sedimentation" and "precipitation" are used synonymously. Similarly, "solubilizer" and "precipitation inhibitor" are used synonymously.

[0017] <Reagent for measuring coagulation time> The reagent for measuring coagulation time according to the first embodiment of the present invention contains ellagic acid and at least one of amino acids and polymers that do not have an aromatic ring, excluding those containing phenol. The following describes embodiments in which the reagent for measuring coagulation time according to this embodiment is an APTT measuring reagent, but the reagent for measuring coagulation time according to this embodiment is not limited to the embodiments described later, and various modifications are possible as long as they do not depart from the gist of the present invention.

[0018] <Ellagic Acid> The ellagic acid is used as a blood coagulation activator in the APTT measurement reagent. The ellagic acid is not particularly limited as long as it is an ellagic acid compound, and examples include ellagic acid, ellagic acid salt, and metal complexes of ellagic acid. The central metal of the metal complex may be an ion or an atom. From the viewpoint of the strength of the activating effect, metal ion complexes of ellagic acid are particularly preferred as the ellagic acid compound. Examples of metal ions include zinc ions, aluminum ions, manganese ions, and copper ions.

[0019] The ellagic acid content in the APTT measurement reagent is not particularly limited, as long as it is an effective amount for activating blood coagulation. The ellagic acid content is preferably 0.001 to 2 mg / mL, more preferably 0.001 to 0.5 mg / mL, even more preferably 0.001 to 0.1 mg / mL, and particularly preferably 0.005 to 0.05 mg / mL, relative to the total volume of the APTT measurement reagent.

[0020] In this embodiment, the reagent may further contain blood coagulation activators other than ellagic acid compounds. Such activators can be any substance that activates contact factors in the intrinsic coagulation pathway, such as kaolin, celite, colloidal silica, polyphenol compounds, anhydrous silicic acid, and metal ions. The amount of activator contained in the APTT measurement reagent is not particularly limited as long as it does not impair the spirit of the present invention, and can be appropriately determined depending on the type of activator and measurement conditions.

[0021] <Amino Acids> The amino acids contained in the APTT measurement reagent of this embodiment are amino acids that do not have an aromatic ring. The type of amino acid is not particularly limited as long as it is effective as a solubilizer for ellagic acid. The amino acid is at least one selected from amino acids found in proteins and their derivatives.

[0022] Examples of amino acids that do not have an aromatic ring include alanine, glycine, arginine, cysteine, proline, threonine, valine, and methionine. Preferably, the amino acid without an aromatic ring is selected from the group consisting of glycine, proline, threonine, valine, and methionine. Examples of derivatives of these amino acids include compounds in which a hydrogen atom or hydroxyl group of the amino acid is substituted with any substituent. The derivative is not particularly limited as long as the substituent does not interfere with the blood coagulation reaction and the solubilization of ellagic acid.

[0023] The amino acid lacking an aromatic ring may be the L-form, the D-form, or a mixture thereof. Furthermore, the amino acid lacking an aromatic ring may be a naturally occurring amino acid or a synthetic amino acid.

[0024] The amino acid content in the APTT measurement reagent is not particularly limited, as long as it is an effective amount for solubilizing ellagic acid. For example, the amino acid content per 100 parts by mass of ellagic acid in the APTT measurement reagent is preferably 20,000 to 270,000 parts by mass, more preferably 22,000 to 270,000 parts by mass.

[0025] The amino acid content in the APTT measurement reagent is preferably 10 to 500 mM, more preferably 20 to 300 mM, and even more preferably 30 to 135 mM. If the amino acid content in the APTT measurement reagent is above the above lower limit, ellagic acid precipitation can be suppressed even when the APTT measurement reagent is stored for a long period of time. The unit "M" for the amino acid content in the APTT measurement reagent indicates the number of moles of amino acids per liter of APTT measurement reagent.

[0026] <Polymers> The polymers included in the APTT measurement reagent are not particularly limited as long as they act as a solubilizer for ellagic acid. Examples of polymers that act as a solubilizer for ellagic acid include bovine serum albumin (Albumin, from Bovine Serum: BSA), Blocking Peptide Fragment (BPF), and chemically synthesized polymers. The polymer included in the APTT measurement reagent is at least one selected from these polymers.

[0027] The BPF is the polypeptide from amino acid positions 419 to 607 of DnaK, a heat shock protein (HSP) derived from Escherichia coli. The BPF may be produced using a known protein expression system, or a commercially available product (for example, Toyobo Co., Ltd., product code: BPF-301, etc.) may be used.

[0028] The aforementioned chemically synthesized polymers are chemically synthesized polymers that do not contain naturally derived building blocks such as natural proteins or natural peptides. Examples of chemically synthesized polymers include, for example, Blockmaster® CE210 and CE510. These are compounds containing polyethylene glycol chains and amino group moieties. The polyethylene glycol chain corresponds to the polymer as defined herein.

[0029] The polymer content in the APTT measurement reagent is not particularly limited, as long as it is an effective amount for solubilizing ellagic acid. For example, the polymer content in the APTT measurement reagent per 100 parts by mass of ellagic acid is preferably 10 to 1,000 parts by mass, more preferably 50 to 500 parts by mass.

[0030] The polymer content in the APTT measurement reagent is preferably, for example, 0.001 to 0.1 mg / mL. If the polymer content in the APTT measurement reagent is above the lower limit mentioned above, ellagic acid precipitation can be suppressed even when the APTT measurement reagent is stored for a long period of time. The polymer content in the APTT measurement reagent indicates the mass of polymer per 1 mL of APTT measurement reagent.

[0031] The solubilizer included in the APTT measurement reagent is at least one selected from the aforementioned amino acids and polymers that do not have an aromatic ring. The inclusion of a solubilizer can suppress the precipitation of ellagic acid.

[0032] Conventional APTT measurement reagents contain phenol as an inhibitor of ellagic acid precipitation. The APTT measurement reagent containing the inhibitor in this embodiment has a higher ability to prevent ellagic acid precipitation than the APTT measurement reagent containing phenol as an inhibitor. Therefore, the APTT measurement reagent in this embodiment does not need to contain phenol as an inhibitor.

[0033] <Metal Salt Compounds> The APTT measurement reagent of this embodiment preferably further contains a metal salt compound. The metal salt compound is at least one metal salt compound selected from the group consisting of manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate, and their hydrates. Copper sulfate and copper chloride are preferred as metal salt compounds to be included in the APTT measurement reagent.

[0034] There are no particular limitations on the content of the metal salt compound in the APTT measurement reagent, and it can be appropriately determined depending on the type of metal salt compound used and the measurement conditions. For example, if the metal salt compound contained in the APTT measurement reagent is copper sulfate, the content of copper sulfate in the APTT measurement reagent is preferably 0.0001 to 0.15 mg / mL, more preferably 0.001 to 0.05 mg / mL, even more preferably 0.005 to 0.04 mg / mL, and particularly preferably 0.0075 to 0.0225 mg / mL, relative to the total volume of the APTT measurement reagent.

[0035] The APTT measurement reagent of this embodiment may further contain phospholipids. Examples of such phospholipids include natural or synthetic phospholipids. Examples of natural phospholipids include phospholipids derived from natural substances such as rabbit brain, bovine brain, human placenta, soybeans, and egg yolk. Examples of synthetic phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), sphingomyelin (SM), and cardiolipin. These phospholipids can be used individually or in combination of two or more. For example, multiple phospholipids can be mixed and used as liposomes.

[0036] There are no particular limitations on the phospholipid content in the APTT measurement reagent, and it can be appropriately determined depending on the type of phospholipid used and the measurement conditions. The phospholipid content in the APTT measurement reagent is preferably 0.005 to 2 mM, more preferably 0.02 to 0.5 mM, relative to the total mass of the APTT measurement reagent.

[0037] The APTT measurement reagent of this embodiment may further contain other additives to improve its shelf life and stability. Such additives may be any additives commonly used in reagents for measuring coagulation time, such as preservatives, antioxidants, and stabilizers. Examples of preservatives include sodium azide and known antibiotics. Examples of antioxidants include butylhydroxyanisole. Examples of stabilizers include polyethylene glycol and polyvinylpyrrolidone.

[0038] <Buffer> The APTT measurement reagent of this embodiment may further contain a buffer. The buffer can be appropriately selected from buffers commonly used in the field of blood testing. Examples of the buffer include PIPES, ACES, HEPES, TAPSO, POPSO, EPPS, and CHES. These buffers can be used individually or in combination of two or more.

[0039] The hydrogen ion concentration of the buffer is not particularly limited as long as it exhibits buffering capacity, and is preferably pH 4 to 9 at 25°C, and more preferably pH 6 to 8. Similarly, the buffer content in the APTT measurement reagent is not particularly limited as long as it exhibits buffering capacity, and is preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, even more preferably 1 to 200 mM, and particularly preferably 10 to 100 mM relative to the total volume of the APTT measurement reagent.

[0040] The APTT measurement reagent of this embodiment may be in liquid form, a frozen form thereof, or a dried form. The dried reagent is dissolved in water or a buffer solution, etc., at the time of use to prepare a liquid reagent.

[0041] The APTT measurement reagent of the present invention may be a reagent kit comprising a first reagent containing an activator and a second reagent containing a coagulation initiator. In the APTT measurement reagent, it is preferable that ellagic acid and at least one of amino acids and polymers that do not have an aromatic ring are contained in the first reagent. The first reagent and the second reagent may be included in the reagent kit individually.

[0042] The second reagent for the APTT assay can contain a component that initiates blood coagulation, such as calcium ions. As the calcium ions, water-soluble calcium compounds such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, and calcium tartrate are used. These calcium compounds can be used either singly or in combination of any two or more. There is no particular limitation on the content of the calcium compound in the APTT assay reagent, and it is preferably 5 to 100 mM, more preferably 10 to 50 mM, based on the total volume of the APTT assay reagent.

[0043] <Method for Measuring APTT> The method for measuring the coagulation time (APTT) according to the second aspect of the present invention includes a step of mixing a blood sample and the reagent for measuring the coagulation time according to the first aspect of the present invention, and a step of measuring the APTT of the sample solution obtained by the above step.

[0044] The blood sample is blood, plasma obtained from blood, or a preparation thereof. In a preferred embodiment of the present invention, there is plasma obtained from blood collected from a subject as the blood sample. The method for obtaining plasma from blood is known in the art. For example, plasma can be obtained by centrifuging blood without hemolyzing it to remove the blood cell components. Further, a known anticoagulant commonly used in clinical tests for blood coagulation ability may be added to the blood collected from the subject. Examples of such an anticoagulant include sodium citrate.

[0045] In the step of mixing the blood sample and the reagent for measuring the coagulation time according to the first aspect of the present invention, first, the blood sample and the first reagent are mixed. The mixing ratio (volume ratio) of the blood sample and the first reagent is preferably 1:1.

[0046] Thereafter, a mixed solution of a blood sample and a first reagent is mixed with a second reagent. The second reagent contains a calcium salt as a coagulation reaction initiator, and mixing the second reagent starts the coagulation reaction. The solution thus obtained may hereinafter be referred to as a sample solution. The final concentrations of ellagic acid and at least one of an amino acid and a polymer in the sample solution are preferably adjusted so as to fall within the preferable ranges described in the <Reagent for Measuring Clotting Time> above.

[0047] Next, the APTT of the sample solution obtained in the above-described step is measured. The APTT may be measured by a conventional method or by an APTT measuring device. There is no particular limitation on the time for measuring the APTT, and it may be from the start point of the coagulation reaction to the point when the end of the coagulation reaction is detected.

[0048] In the measurement by a conventional method, for example, there is a method of visually confirming the precipitation of fibrin in the sample solution and measuring the time from the mixing of the second reagent to the fibrin precipitation.

[0049] The APTT measuring device is not particularly limited as long as it is a known device used in general APTT measurements. When a blood coagulation automatic analyzer (for example, CP3000, manufactured by Sekisui Medical Co., Ltd.) is used as the APTT measuring device, light is irradiated on the measurement sample, the amount of optical change (for example, the amount of change in scattered light intensity) due to the coagulation reaction is measured, and the APTT is calculated from the amount of change.

[0050] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0051] In preparing the reagent for measuring APTT, ellagic acid, copper sulfate pentahydrate, HEPES, liposome, trisodium citrate, procain 300 (manufactured by Sigma-Aldrich, product number: 48914U), calcium chloride, and sodium azide were used.

[0052] The following precipitation inhibitors were used to add to the APTT measurement reagent: phenol, phenylalanine, glycine, proline, threonine, valine, methionine, BSA, BPF (manufactured by TOYOBO, catalog number: BPF-301), and Blockmaster CE210 (manufactured by JSR Life Sciences, catalog number: BLMCE210 (hereinafter sometimes referred to as "CE210")).

[0053] Coagulation time was measured using an optical method. The optical measurement was performed using a CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.), under the following conditions.

[0054] [Conditions for measuring coagulation time] 50 μL of the sample was heated in a cuvette at 37°C for 45 seconds, then 50 μL of the first reagent (R1) was added and heated for a further 171 seconds. 50 μL of the second reagent (R2) was added to the heated mixture to initiate the coagulation reaction. The reaction was carried out at 37°C. The sample (sample solution) to which R2 had been added was irradiated with light of a wavelength of 660 nm, and the amount of optical change (change in scattered light intensity) due to the coagulation reaction was measured. The coagulation time was determined from the measured coagulation reaction.

[0055] 1) The following control samples were used as control specimens: Coagupia Control P-N I (manufactured by Sekisui Medical Co., Ltd.) Coagupia Control P-N II (manufactured by Sekisui Medical Co., Ltd.) Hereinafter, Coagupia Control P-N I may be referred to as "Control I" and Coagupia Control P-N II as "Control II".

[0056] Control I is a control sample with an APTT in the normal range, and Control II is a control sample with an APTT in the abnormal range.

[0057] 2) APTT Measurement Reagents 2-1) Basic Formulation The compositions of the first reagent (R1) and the second reagent (R2) contained in the APTT measurement reagents are shown in Table 1 and Table 2, respectively. The liposomes contained in the first reagent (R1) shown in Table 1 below were prepared according to the method described in International Publication No. 2003 / 015753.

[0058]

[0059]

[0060] 2-2) Test Reagents: • Reagent 1: Basic formula R1 was used. • Reagent 2: Phenol was added to basic formula R1 under the conditions described in Table 3. • Reagents 3-9: Amino acids were added to basic formula R1 under the conditions described in Table 3. • Reagents 10-12: Polymers were added to basic formula R1 under the conditions described in Table 3.

[0061]

[0062] 2-3) Preparation of R1 and R2 The above components were mixed to the predetermined concentration to prepare R1 and R2 of each test reagent, and stored at 4°C. Reagent R1 was also stored separately at 37°C.

[0063] <Test Example 1> 3) Visual Evaluation Three days after R1 preparation, it was visually checked whether ellagic acid precipitation had occurred.

[0064] 3-1) Visual Evaluation Results The results of the visual evaluation are shown in Table 4. For reagents 1 to 4, ellagic acid precipitation was observed under both storage conditions of 4°C and 37°C. Therefore, it was shown that when phenol or phenylalanine, an amino acid having an aromatic ring, is added to R1 in this embodiment as a solubilizer and stored, ellagic acid precipitation occurs.

[0065] On the other hand, no precipitation of ellagic acid was observed in reagents 5 to 12 under either the 4°C or 37°C storage conditions. Therefore, it was shown that when amino acids without aromatic rings (glycine, proline, threonine, valine, and methionine) or polymers (BSA, BPF, and CE210) were added to R1 in this embodiment as solubilizers and stored, precipitation of ellagic acid was suppressed.

[0066] ○: No sedimentation, ×: Sedimentation present

[0067] <Test Example 2> 4) APTT Fluctuation Evaluation The rate of change between APTT immediately after R1 storage and APTT after R1 has been stored for a certain period of time was evaluated.

[0068] 4-1) Heat Treatment: Some reagents were heated to 37°C, higher than room temperature, to allow the reagent components to blend sufficiently with the reagent, and then stored at 37°C. Condition A is the condition without heat treatment, and Condition B is the condition with heat treatment. Reagents in Condition A: Reagents 5, 6, 8, 9, 10. Reagents in Condition B: Reagents 7, 11, 12.

[0069] 4-2) Measurement protocol control I and control II (50 μL each) were heated in a cuvette at 37°C for 45 seconds, then 50 μL of the first reagent (R1) was added and the mixture was heated for a further 171 seconds. 50 μL of the second reagent (R2) was added to the heated mixture to initiate the coagulation reaction, and APTT measurement was performed on the sample immediately after the addition of R2. APTT measurement was performed using an automated blood coagulation analyzer (CP3000, manufactured by Sekisui Medical Co., Ltd.) under the conditions described above.

[0070] R1 was stored at 37°C. Three days after the start of storage of R1, the APTT was calculated for the reagent under condition A using the method described above. Four days after the start of storage of R1, the APTT was calculated for the reagent under condition B using the method described above.

[0071] 4-3) Evaluation Method and Condition A The rate of change in APTT from immediately after the start of R1 storage to 3 days later was calculated using the following formula: APTT rate of change (%) = (APTT 3 days after the start of R1 storage) / (APTT immediately after the start of R1 storage) × 100 Condition B The rate of change in APTT from immediately after the start of R1 storage to 4 days later was calculated using the following formula: APTT rate of change (%) = (APTT 4 days after the start of R1 storage) / (APTT immediately after the start of R1 storage) × 100

[0072] 4-4) APTT Fluctuation Evaluation Results The results of the APTT fluctuation evaluation under condition A are shown in Table 5, and the results of the APTT fluctuation evaluation under condition B are shown in Table 6. From Table 5, it was confirmed that in reagents 5, 6, 8, 9, and 10, in which ellagic acid did not precipitate in the visual evaluation of Test Example 1, the APTT fluctuation rate from immediately after the start of R1 storage was kept within 100 ± 20% in all control samples. Therefore, it was shown that the use of glycine, proline, valine, methionine, and BSA as solubilizers suppressed the precipitation of ellagic acid and also suppressed the fluctuation of APTT.

[0073] Table 6 shows that in the visual evaluation of Test Example 1, reagents 7, 11, and 12, in which ellagic acid did not precipitate, all control samples were found to have their APTT fluctuation rate suppressed to within 100 ± 20% from the start of R1 storage. Therefore, it was shown that the use of threonine, BPF, and CE210 as solubilizers suppressed ellagic acid precipitation and also suppressed APTT fluctuations.

[0074]

[0075]

[0076] According to the present invention, a novel reagent for measuring coagulation time and a method for measuring coagulation time can be provided that can solubilize ellagic acid even without containing phenol.

Claims

1. A reagent for measuring coagulation time that contains ellagic acid and at least one of an amino acid and a polymer that does not have an aromatic ring, and does not contain phenol.

2. The reagent for measuring coagulation time according to claim 1, comprising the amino acid, wherein the content of the amino acid is 20,000 to 270,000 parts by mass per 100 parts by mass of the ellagic acid content.

3. The reagent for measuring coagulation time according to claim 1, comprising the polymer, wherein the content of the polymer is 10 to 1,000 parts by mass per 100 parts by mass of the ellagic acid content.

4. The reagent for measuring coagulation time according to claim 1, comprising the amino acid, wherein the amino acid is at least one amino acid selected from the group consisting of glycine, proline, threonine, valine, and methionine.

5. The reagent for measuring coagulation time according to claim 1, comprising the polymer, wherein the polymer is at least one polymer selected from the group consisting of bovine serum albumin, Blocking Peptide Fragment, and chemically synthesized polymers.

6. The reagent for measuring coagulation time according to claim 1, further comprising a metal salt compound.

7. The reagent for measuring coagulation time according to claim 6, wherein the metal salt compound is at least one metal salt compound selected from the group consisting of manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate, and hydrates thereof.

8. A method for measuring coagulation time, comprising the steps of: obtaining a sample solution by mixing a blood sample with a reagent for measuring coagulation time; and measuring the coagulation time of the sample solution obtained in the first step, wherein the reagent for measuring coagulation time contains ellagic acid and at least one of amino acids and polymers that do not have an aromatic ring, and does not contain phenol.

9. The method for measuring coagulation time according to claim 8, wherein the reagent for measuring coagulation time comprises a first reagent and a second reagent, the first reagent comprises ellagic acid and at least one of an amino acid and a polymer that does not have an aromatic ring, and does not contain phenol, and the second reagent comprises a calcium salt.

10. The method for measuring coagulation time according to claim 9, wherein the step of obtaining the sample solution is to obtain a mixed solution by mixing the blood sample and the first reagent, and then mix the mixed solution with the second reagent.

11. The method for measuring coagulation time according to claim 8, wherein the reagent for measuring coagulation time contains the amino acid, and the content of the amino acid in the reagent for measuring coagulation time is 20,000 to 270,000 parts by mass relative to 100 parts by mass of the ellagic acid content.

12. The method for measuring solidification time according to claim 8, wherein the reagent for measuring solidification time contains the polymer, and the content of the polymer in the reagent for measuring solidification time is 10 to 1,000 parts by mass relative to 100 parts by mass of the ellagic acid content.

13. The method for measuring coagulation time according to claim 8, wherein the reagent for measuring coagulation time contains the amino acid, and the amino acid is at least one amino acid selected from the group consisting of glycine, proline, threonine, valine, and methionine.

14. The method for measuring coagulation time according to claim 8, wherein the reagent for measuring coagulation time comprises the polymer, and the polymer is at least one polymer selected from the group consisting of bovine serum albumin, Blocking Peptide Fragment, and chemically synthesized polymers.

15. The method for measuring coagulation time according to claim 8, wherein the reagent for measuring coagulation time further contains a metal salt compound.

16. The method for measuring solidification time according to claim 15, wherein the metal salt compound is at least one metal salt compound selected from the group consisting of manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate, and hydrates thereof.

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