Reagent and kit for measuring activated partial thromboplastin time, measuring method, and method for inhibiting precipitation of ellagic acid compound

Abstract

The present invention relates to a reagent and a kit for determining the activated partial thromboplastin time, a determination method, and a method for inhibiting the precipitation of ellagic acid compounds. The subject of the present invention is to find other substances with low environmental burden and that can inhibit the precipitation of ellagic acid compounds, and to provide a reagent and a kit for determining the activated partial thromboplastin time (APTT) in which the precipitation of ellagic acid compounds is inhibited. The subject is solved by an APTT determination reagent comprising an ellagic acid compound and a compound represented by the following formula (I) (wherein X is a hydrogen atom, -(C=O)-Y, or -(C=O)-OZ, Y is a hydroxyl group, a hydrogen atom, a linear or branched alkyl group with a carbon number of 1 or more and 6 or less, or a phenyl group, and Z is an alkali metal), and a kit comprising the reagent.

Description

Technical Field

[0001] The present invention relates to a reagent and a kit for measuring activated partial thromboplastin time, a method for measuring activated partial thromboplastin time, and a method for inhibiting the precipitation of ellagic acid compounds. Background Art

[0002] Ellagic acid has the effect of activating the contact factor system, so ellagic acid is often used as an activator of coagulation for the activated partial thromboplastin time (APTT) assay reagent. However, ellagic acid is prone to precipitation in aqueous solvents such as those used in APTT assay reagents. Once the precipitation of ellagic acid is produced, the ellagic acid required for the coagulation reaction is insufficient, and the coagulation time cannot be accurately measured. In the past, as a substance that inhibits the precipitation of ellagic acid, phenol was added to the APTT assay reagent containing ellagic acid. However, phenol is a substance that increases the environmental burden, and the use restrictions on phenol are becoming increasingly strict. Therefore, it is desirable to substantially not contain phenol and the APTT assay reagent in which the precipitation of ellagic acid is suppressed. For example, Patent Document 1 records: as a substance that inhibits the precipitation of ellagic acid, an amino acid with an aromatic ring is added to the APTT assay reagent. Patent Document 2 records: as a substance that inhibits the precipitation of ellagic acid, a polyvinyl alcohol compound is added to the APTT assay reagent.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2013-205087

[0006] Patent Document 2: U.S. Patent Application Publication No. 2017 / 0059594 Summary of the invention

[0007] Problems to be solved by the invention

[0008] The object of the present invention is to find other substances that have low environmental load and can inhibit the precipitation of ellagic acid, and to provide an APTT measurement reagent and a kit in which the precipitation of ellagic acid is inhibited. In addition, the object of the present invention is to provide a method for measuring the APTT time using such substances and a method for inhibiting the precipitation of ellagic acid.

[0009] Solutions to Solve Problems

[0010] The present inventors have found that the precipitation of ellagic acid compounds can be inhibited by using compounds represented by the following formula (I), thereby completing the present invention. Thus, the present invention provides a reagent for measuring activated partial thromboplastin time, comprising an ellagic acid compound and a compound represented by the following formula (I).

[0011]

Chemistry 1

[0012]

[0013] (wherein X is a hydrogen atom, -(C=O)-Y, or -(C=O)-OZ, Y is a hydroxyl group, a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or a phenyl group, and Z is an alkali metal)

[0014] In addition, the present invention provides a kit for determining the activated partial thromboplastin time, which comprises: a first reagent containing an ellagic acid compound and a compound represented by the above formula (I), and a second reagent containing a calcium salt. In addition, the present invention provides a method for determining the activated partial thromboplastin time, which comprises: a step of mixing a blood test sample, a first reagent containing an ellagic acid compound and a compound represented by the above formula (I), and a second reagent containing a calcium salt; and a step of determining the activated partial thromboplastin time of the mixture produced in the step. Further, the present invention provides a method for inhibiting the precipitation of an ellagic acid compound, which inhibits the precipitation of the ellagic acid compound by allowing the ellagic acid compound and the compound represented by the above formula (I) to coexist in an aqueous solution.

[0015] Effects of the Invention

[0016] According to the present invention, it is possible to provide an APTT measurement reagent and a kit that suppresses the precipitation of ellagic acid without containing phenol, which is a substance that imposes a heavy environmental load. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram showing an example of the APTT reagent according to the present embodiment.

[0018] Figure 2 This is a schematic diagram showing an example of the APTT kit according to the present embodiment.

[0019] Figure 3 It is a graph showing the relationship between the particle size of the complex and the sodium chloride concentration in ellagic acid metal ion complex solutions containing various additives.

[0020] Figure 4A This is a graph showing the relationship between the particle size of the complex and the sodium chloride concentration in ellagic acid metal ion complex solutions containing gallic acid at various concentrations.

[0021] Figure 4B This is a graph showing the relationship between the particle size of the complex and the sodium chloride concentration in ellagic acid metal ion complex solutions containing pyrogallol at various concentrations.

[0022] Figure 5 The figure shows the coagulation waveform obtained by optical measurement using an APTT reagent containing gallic acid or pyrogallol. DETAILED DESCRIPTION

[0023] [1. Reagents for determination of activated partial thromboplastin time]

[0024] The APTT reagent of the present embodiment contains a compound represented by the following formula (I) as a substance that inhibits the precipitation of ellagic acid.

[0025]

Chemistry 2

[0026]

[0027] (wherein X is a hydrogen atom, -(C=O)-Y, or -(C=O)-OZ, Y is a hydroxyl group, a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or a phenyl group, and Z is an alkali metal)

[0028] In formula (I), as the linear or branched alkyl group having 1 to 6 carbon atoms, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, isopentyl (isoamyl), isohexyl and the like groups can be cited. When X in formula (I) is -(C=O)-OZ, formula (I) represents an alkali metal salt of gallic acid. As the alkali metal, for example, sodium, potassium, lithium and the like can be cited. Among these, sodium and potassium are preferred.

[0029] Examples of the compound represented by the formula (I) include pyrogallol, gallic acid, 3,4,5-trihydroxybenzaldehyde, methyl gallate, ethyl gallate, propyl gallate, butyl gallate, amyl gallate, hexyl gallate, isopropyl gallate, isobutyl gallate, isoamyl gallate, isohexyl gallate, benzopyrogallol (3,4,5-trihydroxybenzophenone), sodium gallate, potassium gallate, etc. Among these, pyrogallol and gallic acid are preferred.

[0030] The content of the compound shown in formula (I) in the APTT reagent can be determined according to the concentration of the ellagic acid compound in the reagent. The concentration of the ellagic acid compound in the APTT reagent is as long as it is the degree commonly used in the APTT assay, and the content of the compound shown in formula (I) in the APTT reagent is, for example, 0.001% by mass or more and 0.02% by mass or less, preferably 0.005% by mass or more and 0.02% by mass or less. In a preferred embodiment, the content of the compound shown in formula (I) in the APTT reagent is 5 parts by mass or more and 65 parts by mass or less relative to 100 parts by mass of ellagic acid compounds, more preferably 20 parts by mass or more and 65 parts by mass or less.

[0031] The APTT reagent of the present embodiment includes an ellagic acid compound as an activator. The ellagic acid compound can be any one of ellagic acid, ellagic acid salts, and metal complexes of ellagic acid. The central metal of the metal complex can be an ion or an atom. Between the metal complexes of ellagic acid, the central metal can be the same or different. From the viewpoint of the intensity of the activation effect, as the ellagic acid compound, the metal ion complex of ellagic acid is particularly preferred. As metal ions, for example, zinc ions, aluminum ions, manganese ions, etc. can be cited. The concentration of the ellagic acid compound in the APTT reagent is usually 10 μM or more and 400 μM or less, preferably 30 μM or more and 150 μM or less.

[0032] In this embodiment, an activator other than ellagic acid compounds may be further included. Such activators may be substances that activate contact factors in the intrinsic coagulation pathway, and examples thereof include silicon dioxide, kaolin, diatomaceous earth, etc. For example, when the activator is silicon dioxide, the content of the activator in the APTT reagent is usually 0.1 mg / mL or more and 1.0 mg / mL or less, preferably 0.2 mg / mL or more and 0.6 mg / mL or less.

[0033] The APTT reagent of the present embodiment preferably contains phospholipids. Examples of phospholipids include phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), etc. The APTT reagent of the present embodiment preferably contains at least one selected from the group consisting of PE, PC and PS, and more preferably contains PE, PC and PS. The phospholipids may be phospholipids of natural origin or synthetic phospholipids. Examples of phospholipids of natural origin include phospholipids from rabbit brain, bovine brain, human placenta, soybeans, egg yolks, etc. Synthetic phospholipids or phospholipids of natural origin purified to a purity of 99% or more are particularly preferred.

[0034] The fatty acid side chains (acyl groups) of PE, PC and PS are not particularly limited, and examples thereof include acyl groups having a carbon number of 8 or more and 20 or less, preferably acyl groups having a carbon number of 14 or more and 18 or less. Examples of such acyl groups include lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl and the like. PE, PC and PS all have two fatty acid side chains in the molecule. The two fatty acid side chains in each molecule of PE, PC and PS may be the same or different.

[0035] As specific PE, for example, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, etc. can be cited. As specific PC, for example, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, etc. can be cited. As specific PS, for example, dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dioleoylphosphatidylserine, etc. can be cited.

[0036] The concentration of phospholipids in the APTT reagent can be appropriately determined according to the type of phospholipids and the measurement conditions. For example, the concentration of phospholipids in the APTT reagent is 30 to 2000 μg / mL, preferably 60 to 1000 μg / mL. When the APTT reagent contains PE, PC and PS as phospholipids, in the reagent, the concentration of PE is usually 10 to 700 μg / mL, preferably 30 to 300 μg / mL, the concentration of PC is usually 20 to 1000 μg / mL, preferably 30 to 500 μg / mL, and the concentration of PS is usually 3 to 300 μg / mL, preferably 5 to 150 μg / mL.

[0037] The APTT reagent of the present embodiment preferably contains metal ions. By containing metal ions, the activation of contact factors and the effect of inhibiting the generation of precipitates can be expected. In addition, the ellagic acid in the reagent forms a chelate with the metal ions generated by the metal ion-forming compound to form a metal ion complex of ellagic acid. The APTT reagent containing metal ions can be prepared by adding the metal ion-forming compound to the APTT reagent. The metal ion-forming compound is a compound that ionizes in an aqueous solution to produce metal ions. The metal ion-forming compound is not particularly limited as long as it produces metal ions in the APTT reagent and the anions produced by the compound do not inhibit the blood coagulation reaction. As the metal ion-forming compound, for example, salts of metals and organic acids or inorganic acids can be listed. Among these, salts of metals and inorganic acids are preferred, and salts of acids such as hydrochloric acid, sulfuric acid, and nitric acid and metals can be listed. More preferred metal salts are salts of at least one metal selected from zinc salts, manganese salts, aluminum salts, and nickel salts. The metal ion-forming compound can be one, or two or more can be combined. In a preferred embodiment, the metal ion forming compound is a mixture of a zinc ion forming compound and an aluminum ion forming compound, or a mixture of a manganese ion forming compound and an aluminum ion forming compound. Examples of such compounds include zinc chloride, manganese chloride, aluminum chloride, nickel chloride, etc. The metal ion forming compound may be an anhydrous substance or a hydrate. The concentration of the metal ion in the APTT reagent is, for example, 1 μM or more and 1 mM or less, preferably 10 μM or more and 500 μM or less.

[0038] In order to improve stability, the APTT reagent of the present embodiment may further include non-polar amino acids. As non-polar amino acids, for example glycine, alanine, phenylalanine, etc. can be cited. Glycine also acts as a buffer, and phenylalanine also acts as a precipitation inhibitor of ellagic acid. Therefore, as non-polar amino acids, glycine and phenylalanine are preferred. The non-polar amino acid can be any of the L-body, the D-body and their mixture. The non-polar amino acid can be an amino acid of natural origin or a synthetic amino acid. The concentration of the non-polar amino acid in the APTT reagent can be appropriately set, usually 0.75 mass % or more, preferably 0.75 mass % or more and 5 mass % or less, more preferably 0.75 mass % or more and 1.5 mass % or less.

[0039] The APTT reagent of the present embodiment may further include additives for improving preservation and stability. Examples of such additives include preservatives, antioxidants, stabilizers, and the like. Examples of preservatives include antibiotics such as aminoglycoside antibiotics, sodium azide, and the like. Commercially available preservatives such as ProClin (trademark) 300 may be added. Examples of antioxidants include butylated hydroxyanisole, and the like. Examples of stabilizers include polyethylene glycol, polyvinyl pyrrolidone, and the like.

[0040] The solvent used in the APTT reagent of the present embodiment can be appropriately selected from aqueous solvents commonly used in the field of blood tests. As such aqueous solvents, water, physiological saline, buffer solutions, etc. can be cited, for example. The pH of the buffer solution is preferably above 6 and below 8, more preferably above 7 and below 7.6. As buffer solutions, GOOD'S buffer solutions such as HEPES, TAPS, MOPS, BES, TES, Tris hydrochloric acid buffer solutions (Tris-HCl), Owren's veronal buffer solutions, imidazole hydrochloric acid buffer solutions, etc. can be cited. The buffer contained in the buffer solution can be one or more than two. Glycine can be added to the buffer solution as needed.

[0041] The pH of the APTT reagent of the present embodiment is usually 6 to 8, preferably 7 to 7.6. The pH of the APTT reagent can be adjusted by adding the above-mentioned buffer.

[0042] The APTT reagent of this embodiment can not only prevent the precipitation of ellagic acid, but also reduce the initial reaction abnormality in the optical measurement of APTT by including the compound shown in the above formula (I). The initial reaction abnormality refers to the phenomenon that the optical measurement value of the test subject changes from the moment the APTT reagent is added to the blood test subject. When a normal blood test subject is mixed with the APTT reagent, the optical measurement value of the test subject usually remains almost unchanged from the addition of the reagent to the precipitation of fibrin (the beginning of coagulation), and changes significantly from the precipitation of fibrin. However, initial reaction abnormalities sometimes occur in blood samples of patients with coagulation abnormalities. Depending on the measuring device, there is a situation where the correct coagulation time cannot be obtained when the initial reaction abnormality is detected.

[0043] The APTT reagent of the present embodiment can be manufactured in the same manner as the manufacturing method of the conventional APTT reagent, except that the compound shown in the above formula (I) is used instead of phenol as the precipitation inhibitor of ellagic acid. As an example, the preparation of the APTT reagent of the present embodiment comprising phospholipids is described. First, ellagic acid or ellagic acid salt is dissolved in an aqueous solvent to prepare an aqueous solution of ellagic acid. As an aqueous solvent, GOOD'S buffer is preferably used. Metal ions are added to the obtained ellagic acid solution to form a compound, thereby preparing a solution of a metal ion complex comprising ellagic acid. The above-mentioned phospholipids are added to the obtained solution, thereby obtaining the APTT reagent of the present embodiment comprising phospholipids. As required, the above-mentioned non-polar amino acids, preservatives, antioxidants, stabilizers, etc. can be added.

[0044] The APTT reagent of the present embodiment can be used under conventional APTT assay conditions in the same manner as conventional APTT reagents. In addition, the APTT reagent of the present embodiment can be used for screening tests of endogenous coagulation factors, monitoring of heparin therapy, and screening tests of lupus anticoagulants (LA) in the same manner as conventional APTT reagents. The mixing ratio of the APTT reagent of the present embodiment to the blood specimen can be about 8:2 to 2:8, preferably 5:5, expressed as a volume ratio. In other words, the amount of the APTT reagent added of the present embodiment can be an amount (volume) greater than 0.25 times and less than 4 times the amount (volume) of the blood specimen. Preferably, the amount of the APTT reagent added of the present embodiment is equal to the amount (volume) of the blood specimen.

[0045] When the APTT reagent of the present embodiment is used to measure the coagulation time of a normal subject, a measurement result equivalent to that of the conventional APTT reagent can be obtained. For example, when the APTT reagent of the present embodiment is appropriately used together with an aqueous solution containing calcium ions and the APTT of a specified normal plasma (e.g., Coragra Testor IX manufactured by Sysmex Corporation) is measured, the APTT is expected to be about 30 seconds (e.g., more than 25 seconds and less than 35 seconds). In addition, when the APTT of a specified abnormal plasma (e.g., Coragra Testor IIX manufactured by Sysmex Corporation) is measured, the APTT is expected to be within the range of 60 to 100 seconds.

[0046] In this embodiment, the container containing the APTT reagent can be packaged into a box and provided to the user. The box can also contain an additional document describing the use of the APTT reagent. Figure 1 An example of the APTT reagent according to this embodiment is shown. Figure 1 , 10 denotes a container containing the APTT reagent of the present embodiment, 11 denotes a packaging box, and 12 denotes an attached document.

[0047] [2. Kit for assaying activated partial thromboplastin time]

[0048] The APTT assay kit of the present embodiment (hereinafter also referred to as "kit") comprises: a first reagent containing an ellagic acid compound and a compound represented by the above formula (I), and a second reagent containing a calcium salt. As the first reagent, the APTT reagent of the present embodiment described above can be used. The details of the first reagent are the same as those described for the APTT reagent of the present embodiment. The details of the ellagic acid compound are as described above.

[0049] The second reagent is a reagent that is added to a mixture of a blood sample and the first reagent and that triggers blood coagulation. The second reagent is preferably an aqueous solution containing calcium ions. As the aqueous solution containing calcium ions, an aqueous solution of a calcium salt is preferred. As the calcium salt, for example, calcium chloride can be cited. The calcium ion concentration in the second reagent is generally 2.5 mM or more and 40 mM or less, preferably 10 mM or more and 30 mM or less. When a calcium salt that is easily soluble in water, such as calcium chloride, is used, the calcium ion concentration in the second reagent can be expressed as the concentration of the calcium salt.

[0050] In this embodiment, the container containing the first reagent and the container containing the second reagent can be packaged into a box and provided to the user. The box can also contain an additional document describing the use of the APTT kit. Figure 2 An example of the APTT kit according to this embodiment is shown. Figure 2, 20 denotes a reagent kit of the present embodiment, 21 denotes a first container containing a first reagent, 22 denotes a second container containing a second reagent, 23 denotes a packaging box, and 24 denotes an additional document.

[0051] The kit of the present embodiment may further include reagents commonly used for APTT determination as other reagents. Examples of such reagents include reference plasma and aqueous media for dilution. Examples of reference plasma include normal plasma, plasma for precision management, plasma lacking various coagulation factors, plasma containing LA, plasma containing heparin, and the like. The aqueous medium for dilution is an aqueous medium for diluting the first reagent. For example, when detecting LA using the kit of the present embodiment having a first reagent containing phospholipids, the first reagent is diluted with an aqueous medium for dilution to reduce the phospholipid concentration of the first reagent. As a result, the phospholipid inhibition reaction caused by LA is more easily expressed. In the field of blood testing, the APTT measured by the diluted first reagent and the second reagent is referred to as "diluted APTT" (dAPTT).

[0052] [3. Method for inhibiting precipitation of ellagic acid compound]

[0053] Another embodiment provides a method for inhibiting the precipitation of ellagic acid compounds. In this method, the precipitation of ellagic acid compounds is inhibited by allowing ellagic acid compounds and the compound represented by the above formula (I) to coexist in an aqueous solution. The details of the ellagic acid compound and the compound represented by formula (I) are as described above. The aqueous solution can be an aqueous solvent that can dissolve the ellagic acid compound and the compound represented by formula (I). The details of this type of aqueous solvent are the same as the details described for the aqueous solvent used in the APTT reagent of this embodiment.

[0054] The method of making the ellagic acid compound and the compound shown in the above formula (I) coexist in an aqueous solution is not particularly limited. For example, the ellagic acid compound can be added to an aqueous solution containing the compound shown in formula (I). In addition, the compound shown in formula (I) can be added to an aqueous solution containing the ellagic acid compound. Alternatively, the aqueous solution containing the compound shown in formula (I) can be mixed with the aqueous solution containing the ellagic acid compound.

[0055] As shown in the examples described below, for an aqueous solution of an ellagic acid compound that does not contain a compound represented by formula (I), if the salt concentration in the aqueous solution is increased, precipitation of the ellagic acid compound occurs due to the salting-out effect. However, when an ellagic acid compound and a compound represented by formula (I) are allowed to coexist in an aqueous solution, precipitation of the ellagic acid compound is suppressed even if the salt concentration in the aqueous solution is increased.

[0056] [4. Method for measuring activated partial thromboplastin time]

[0057] The APTT determination method of the present embodiment (hereinafter also referred to as "determination method") comprises the following steps: mixing a blood test subject, a first reagent containing an ellagic acid compound and a compound represented by the above formula (I), and a second reagent containing calcium ions, and determining the clotting time. As the first reagent, the APTT reagent of the present embodiment described above can be used. Alternatively, as the first reagent and the second reagent, the kit of the present embodiment described above can be used.

[0058] As the blood specimen, blood collected from a subject or plasma prepared from the blood is used. The preferred blood specimen is plasma. A known anticoagulant commonly used in blood tests may be added to the blood specimen. Examples of such anticoagulants include trisodium citrate. When monitoring heparin therapy by the measurement method of this embodiment, blood or plasma from a subject to whom heparin has been administered is used. Before adding the first reagent, the blood specimen may be incubated for a period of time of 30 seconds to 2 minutes at a temperature of, for example, 35°C to 40°C.

[0059] In the present embodiment, a blood specimen is first mixed with a first reagent. The mixing ratio of the first reagent to the blood specimen can be about 8:2 to 2:8, preferably 5:5, expressed as a volume ratio. In other words, the amount of the first reagent added can be an amount (volume) that is greater than 0.25 times and less than 4 times the amount (volume) of the blood specimen. Preferably, the amount of the first reagent added is equal to the amount (volume) of the blood specimen. After the blood specimen and the first reagent are mixed, the mixture is preferably incubated under prescribed conditions. As prescribed conditions, for example, the following conditions can be cited: incubation at a temperature of greater than 35°C and less than 40°C for a time of greater than 2 minutes and less than 5 minutes.

[0060] Next, the blood sample is mixed with the mixture of the first reagent and the second reagent. Hereinafter, the mixture of the blood sample, the first reagent and the second reagent is also referred to as the "measurement sample". Regarding the amount of the second reagent added, it is sufficient to make the calcium ion concentration in the measurement sample reach an amount (volume) of usually 2 mM to 20 mM, preferably 4 mM to 10 mM. The preparation of the measurement sample can be carried out manually or by a fully automatic measurement device. Examples of such devices include the CS series of fully automatic blood coagulation measurement devices (Sysmex Corporation) and the like.

[0061] In the present embodiment, the moment of adding the second reagent is used as the starting point of the measurement, and the coagulation time is measured. The coagulation time can be measured by a manual method or by a fully automatic measuring device. In the manual method, a stopwatch or the like is used to visually measure the time until fibrin precipitation. When using a fully automatic measuring device, the coagulation time can be measured by an optical measurement method or by a physical measurement method. In the optical measurement method, for example, light is irradiated on the measurement sample to obtain optical information related to transmittance, absorbance, scattered light intensity, etc., and the coagulation time is obtained based on the obtained information. In the physical measurement method, for example, a steel ball is used to obtain physical information related to the viscosity of the measurement sample, and the coagulation time is obtained based on the obtained information. The fully automatic measuring device is not particularly limited. For example, the CS series (Sysmex Corporation) of the fully automatic blood coagulation measuring device can measure the coagulation time based on optical information such as transmittance, absorbance, and scattered light intensity. The fully automatic blood coagulation and fibrinolysis measuring device STA Compact (Rosshu Diagnosis Technology Co., Ltd.) can measure the coagulation time based on physical information such as viscosity.

[0062] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples.

[0063] [Example]

[0064] Example 1: Study on the inhibitory effect of ellagic acid compounds on precipitation

[0065] In the APTT reagent containing ellagic acid metal ion complex as an activator, when the salt concentration increases, the complex particle size increases due to the salting-out effect, and finally precipitation occurs. Using this salting-out principle, the precipitation inhibition effect of various additives in ellagic acid metal ion complex solutions was confirmed.

[0066] (1) Preparation of Ellagic Acid Metal Ion Complex Solution

[0067] An ellagic acid metal ion complex solution was prepared containing 50 mM HEPES, 8 mM TAPS and 1 mass % glycine as buffer components, 100 μM ellagic acid in terms of the metal ion complex of ellagic acid, 60 μM zinc chloride and 50 μM aluminum chloride, and 0.05% ProClin (trademark) 300 (Sigma-Aldrich) as a preservative. The solution was mixed with at least one selected from gallic acid, pyrogallol, hydroquinone, 5-methylpyrogallol, resorcinol, and cyclohexanetriol as an additive to obtain a complex solution containing each additive at a concentration of 100 μM. As a control, an ellagic acid metal ion complex solution without additives was used.

[0068] (2) Study on precipitation inhibition effect

[0069] Each complex solution prepared in (1) above was mixed with a 5M sodium chloride solution to obtain a mixed solution (test area) having a sodium chloride concentration of 10 mM, 50 mM, or 100 mM. The relationship between the salt concentration of each mixed solution and the average particle size of the complex was confirmed by the dynamic scattered light method using a Zetasizer Nano (Malvern). The results are shown in Figure 3 .like Figure 3 As shown in the figure, it can be seen that the increase in particle size in the test area with gallic acid or pyrogallol added is small relative to the increase in salt concentration. In fact, when the sodium chloride concentration is 100mM, although no precipitation is visually confirmed in the test area with gallic acid or pyrogallol added, precipitation is visually confirmed in the test area containing other additives and the test area without additives.

[0070] (3) Study on the relationship between additive concentration and precipitation inhibition effect

[0071] Gallic acid and pyrogallol, which have the effect of inhibiting the precipitation of complex particles, are used to achieve Figure 4A and 4B The concentrations shown were added to the ellagic acid metal ion complex solution. The precipitation inhibition effect was confirmed in the same manner as in (2) above using complex solutions containing gallic acid or pyrogallol at various concentrations. The results are shown in Figure 4A and B. Show: The precipitation inhibition effect is visible at any concentration.

[0072] Example 2: Study on the effect of suppressing abnormal initial reaction of coagulation

[0073] Whether the abnormal initial reaction can be suppressed was investigated by using an APTT reagent containing pyrogallol or gallic acid.

[0074] (1) Preparation of APTT reagent

[0075] An ellagic acid metal ion complex solution was prepared containing 50 mM HEPES, 8 mM TAPS and 1 mass % glycine as buffer components, 100 μM ellagic acid, 60 μM zinc chloride and 50 μM aluminum chloride as metal ion complex of ellagic acid, and 0.05% ProClin (trademark) 300 (Sigma-Aldrich) as a preservative. The complex solution was mixed with gallic acid or pyrogallol as an additive to obtain a complex solution containing gallic acid or pyrogallol at a concentration of 100 μM. As a control, an ellagic acid metal ion complex solution without additives was used. As phospholipid components, PE (L-α-phosphatidylethanolamine), PC (L-α-phosphatidylcholine) and PS (L-α-phosphatidylserine) were added to each complex solution in a manner to achieve a final concentration of 10 mg / dL, 45 mg / dL and 5 mg / dL, respectively, to prepare an APTT reagent. The contents of gallic acid and pyrogallol in each APTT reagent were 0.013% by mass and 0.009% by mass, respectively. In addition, the contents of gallic acid and pyrogallol in each APTT reagent were 43 parts by mass and 32 parts by mass per 100 parts by mass of ellagic acid, respectively.

[0076] (2) Research on the inhibitory effect of initial abnormal reaction

[0077] Very low density lipoprotein (VLDL) and C-reactive protein (CRP) were added to frozen plasma to a final concentration of 100 μg / mL and 300 μg / mL, respectively, to prepare a simulated sample that induces abnormal initial coagulation reaction. The simulated sample was subjected to APTT measurement using a fully automatic coagulation measurement apparatus CS-5100 (Sysmex Corporation) equipped with the reagent prepared in (1) above and a reagent containing calcium ions, and its coagulation waveform was obtained. Figure 5 The coagulation waveforms obtained by measurement using each APTT reagent are shown.

[0078] like Figure 5 As shown, in the control area (APTT reagent without additives), the optical measurement value immediately after the reagent was added decreased sharply, and abnormal initial reaction of coagulation was observed. On the other hand, in the test area using APTT reagent containing gallic acid or pyrogallol, the decrease in the optical measurement value after the reagent was added became gentle, and it can be confirmed that abnormal initial reaction of coagulation was reduced.

[0079] Explanation of symbols

[0080] 10: Container for storing the APTT reagent of this embodiment

[0081] 20: Kit of this embodiment

[0082] 21: Container 1

[0083] 22: Container 2

[0084] 11, 23: Packing box

[0085] 12, 24: Attach files

Claims

1. A reagent for measuring activated partial thromboplastin time, comprising as an active ingredient: Ellagic acid compounds, and At least one compound selected from the group consisting of pyrogallol and gallic acid.

2. The reagent according to claim 1, wherein The content of at least one compound selected from the group consisting of pyrogallol and gallic acid is 0.001 mass % or more and 0.02 mass % or less.

3. The reagent according to claim 1, wherein The content of the at least one compound selected from the group consisting of pyrogallol and gallic acid is 5 parts by mass or more and 65 parts by mass or less relative to 100 parts by mass of the ellagic acid compound. The reagent according to claim 1 , further comprising a metal ion.

5. The reagent according to claim 4, wherein The metal ions are a mixture of zinc ions and aluminum ions, or a mixture of manganese ions and aluminum ions. The reagent according to claim 1 , further comprising a non-polar amino acid.

7. The reagent according to claim 6, wherein The non-polar amino acid is at least one selected from the group consisting of glycine, alanine, and phenylalanine.

8. The reagent according to claim 6 or 7, wherein The content of the non-polar amino acid is 0.75% by mass or more.

9. The agent according to claim 1, further comprising a phospholipid.

10. The reagent according to claim 9, wherein The phospholipid is at least one selected from the group consisting of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine.

11. A kit for measuring activated partial thromboplastin time, comprising as an active ingredient: a first agent comprising an ellagic acid compound and at least one compound selected from the group consisting of pyrogallol and gallic acid, and The second reagent comprises a calcium salt.

12. The kit according to claim 11, wherein The content of at least one compound selected from the group consisting of pyrogallol and gallic acid is 0.001 mass % or more and 0.02 mass % or less.

13. The kit according to claim 11, wherein The content of the at least one compound selected from the group consisting of pyrogallol and gallic acid is 5 parts by mass or more and 65 parts by mass or less relative to 100 parts by mass of the ellagic acid compound. The kit according to claim 11 , wherein the first reagent further comprises metal ions.

15. The kit according to claim 14, wherein The metal ions are a mixture of zinc ions and aluminum ions, or a mixture of manganese ions and aluminum ions. The kit according to claim 11 , wherein the first reagent further comprises a non-polar amino acid.

17. The kit according to claim 16, wherein The non-polar amino acid is at least one selected from the group consisting of glycine, alanine, and phenylalanine.

18. The kit according to claim 16 or 17, wherein The content of the non-polar amino acid is 0.75% by mass or more. The kit according to claim 11 , wherein the first reagent further comprises a phospholipid.

20. The kit according to claim 19, wherein The phospholipid is at least one selected from the group consisting of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine.

21. A method for determining activated partial thromboplastin time, comprising: A step of mixing a blood sample, a first reagent containing an ellagic acid compound and at least one compound selected from the group consisting of pyrogallol and gallic acid, and a second reagent containing a calcium salt; and A step of measuring the activated partial thromboplastin time of the mixture produced in the above step.

22. The method according to claim 21, wherein: The content of at least one compound selected from the group consisting of pyrogallol and gallic acid is 0.001 mass % or more and 0.02 mass % or less.

23. The method according to claim 21, wherein: The content of the at least one compound selected from the group consisting of pyrogallol and gallic acid is 5 parts by mass or more and 65 parts by mass or less relative to 100 parts by mass of the ellagic acid compound.

24. The method according to claim 21, wherein the first reagent further comprises a metal ion.

25. The method according to claim 24, wherein: The metal ions are a mixture of zinc ions and aluminum ions, or a mixture of manganese ions and aluminum ions.

26. The method of claim 21, wherein the first reagent further comprises a non-polar amino acid.

27. The method according to claim 26, wherein: The non-polar amino acid is at least one selected from the group consisting of glycine, alanine, and phenylalanine.

28. The method according to claim 26 or 27, wherein: The content of the non-polar amino acid is 0.75% by mass or more.

29. The method of claim 21, wherein the first reagent further comprises a phospholipid.

30. The method of claim 29, wherein: The phospholipid is at least one selected from the group consisting of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine.

31. A method for inhibiting precipitation of ellagic acid compounds, wherein: The precipitation of the ellagic acid compound is suppressed by allowing the ellagic acid compound and at least one compound selected from the group consisting of pyrogallol and gallic acid to coexist in the aqueous solution.

32. The method according to claim 31, wherein: The content of at least one compound selected from the group consisting of pyrogallol and gallic acid is 0.001 mass % or more and 0.02 mass % or less.

33. The method according to claim 31, wherein: The content of the at least one compound selected from the group consisting of pyrogallol and gallic acid is 5 parts by mass or more and 65 parts by mass or less relative to 100 parts by mass of the ellagic acid compound.

34. The method of claim 31, wherein the aqueous solution further comprises metal ions.

35. The method of claim 34, wherein: The metal ions are a mixture of zinc ions and aluminum ions, or a mixture of manganese ions and aluminum ions.

36. The method of claim 31, wherein the aqueous solution further comprises a non-polar amino acid.

37. The method of claim 36, wherein: The non-polar amino acid is at least one selected from the group consisting of glycine, alanine, and phenylalanine.

38. The method according to claim 36 or 37, wherein: The content of the non-polar amino acid is 0.75% by mass or more.

39. The method of claim 31, wherein the aqueous solution further comprises a phospholipid.

40. The method of claim 39, wherein: The phospholipid is at least one selected from the group consisting of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine.

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