JP2013205087A - Reagent for coagulation time measurement, reagent kit for coagulation time measurement and coagulation time measuring method - Google Patents

Abstract

【Task】
An object of the present invention is to provide a reagent for measuring a coagulation time and a reagent kit that can prevent precipitation of ellagic acid without using phenol. Another object of the present invention is to provide a coagulation time measurement method using such a reagent and a reagent kit.
[Solution]
The reagent for measuring the coagulation time and the reagent kit include ellagic acid as an activator and an amino acid having an aromatic ring.
[Selection figure] None

Description

  The present invention relates to a coagulation time measurement reagent, a coagulation time measurement reagent kit, and a coagulation time measurement method used in a clinical test for blood coagulation ability.

  In the field of clinical examination, a test for examining an abnormality of a blood coagulation factor in a subject by measuring a time required for blood coagulation (hereinafter also referred to as “coagulation time”) is known. One common laboratory test for blood clotting is the measurement of activated partial thromboplastin time (APTT). APTT is a clotting time that reflects the function of the intrinsic clotting pathway that is initiated by the blood contacting a foreign substance having a negative charge during bleeding. APTT measurement is used for screening for deficiencies and abnormalities in endogenous clotting factors, monitoring for heparin therapy, and the like.

  A general technique has been established for the measurement of APTT, using an activator having a negative charge, a phospholipid that is an alternative to platelet factor 3 and a calcium salt to supply calcium ions. Measure APTT. The measurement principle is as follows. First, the contact factor system in plasma is activated by the activator. When a calcium salt is added here, the coagulation reaction is promoted by the action of the activated contact factor system and phospholipid, and fibrin is finally deposited in plasma. In the APTT measurement, fibrin precipitation is regarded as coagulation, and the coagulation time until then is measured.

  The above APTT measurement is also used to detect lupus anticoagulant (LA). LA is a kind of antibody responsible for antiphospholipid syndrome. It is known that when LA is present in the blood of a subject, clotting time is prolonged because LA inhibits phospholipids necessary for blood clotting. By confirming the prolongation of the coagulation time by measuring APTT, it becomes possible to detect LA in plasma.

  So far, various reagents that enable the measurement of the coagulation time as described above have been marketed. Such reagents include the activators described above to promote blood clotting in vitro. As activating agents, ellagic acid, celite, kaolin, etc. are known. Among them, ellagic acid shows a strong activation action by forming a chelate with a metal ion, so a reagent for measuring the coagulation time It is often used for.

  However, since ellagic acid has a property that it is difficult to dissolve in an aqueous medium usually used for a reagent for measuring a coagulation time, there is a problem that ellagic acid is likely to precipitate in a reagent containing ellagic acid. If precipitation of ellagic acid occurs, the amount of ellagic acid used for the coagulation reaction is insufficient, so that the progress of the coagulation reaction is delayed, and as a result, the coagulation time is extended. In order to solve such a problem, Patent Document 1 discloses that ellagic acid is solubilized in a reagent using phenol. At present, it is the mainstream to add phenol as a precipitation inhibitor to a reagent containing ellagic acid as an activator.

  However, since restrictions on the use of phenol, which is an environmentally hazardous substance, are becoming stricter, it is desired to avoid the use of phenol even in trace amounts.

  Patent Document 2 discloses a technique in which a reagent for measuring APTT contains alanine, glycine, and valine in order to improve the stability of the reagent. However, since Patent Document 2 does not consider at all the suppression of precipitation of ellagic acid, the reagent disclosed in Patent Document 2 may cause precipitation of ellagic acid.

Japanese National Patent Publication No. 5-506309 Japanese Patent Application Laid-Open No. 7-151763

  In view of the above circumstances, an object of the present invention is to provide a coagulation time measurement reagent and a reagent kit that can prevent precipitation of ellagic acid without using phenol. Another object of the present invention is to provide a coagulation time measurement method using such a reagent and a reagent kit.

  By adding an amino acid having an aromatic ring to a reagent for measuring a coagulation time containing ellagic acid as an activator, the present inventors can prevent precipitation of ellagic acid and affect the measurement of the coagulation time. As a result, the present invention was completed.

  Therefore, according to the present invention, a clotting time measurement reagent containing ellagic acid as an activator and an amino acid having an aromatic ring is provided.

  In addition, according to the present invention, there is provided a reagent kit for coagulation time measurement comprising ellagic acid as an activator, a first reagent containing an amino acid having an aromatic ring, and a second reagent containing a calcium salt. .

Furthermore, according to the present invention,
A first mixing step of mixing a blood sample with ellagic acid as an activator and a first reagent containing an amino acid having an aromatic ring;
A second mixing step of mixing the sample obtained in the first mixing step with a second reagent containing a calcium salt;
And a step of measuring the coagulation time of the sample obtained in the second mixing step.

According to the coagulation time measurement reagent and the reagent kit of the present invention, it becomes possible to prevent precipitation of ellagic acid by using an amino acid having an aromatic ring. There is no need to add the substance phenol to the reagent.
Furthermore, the coagulation time measurement reagent and reagent kit of the present invention have the same level of measurement ability as conventional coagulation time measurement reagents containing phenol. Therefore, the coagulation time measurement method of the present invention using the coagulation time measurement reagent and reagent kit of the present invention can measure the coagulation time of a blood sample in the same manner as the measurement method using a conventional reagent.

[1] Reagent for measuring clotting time The reagent for measuring clotting time of the present invention (hereinafter also simply referred to as “reagent”) comprises ellagic acid as an activator and an aromatic ring as an ellagic acid precipitation inhibitor. Including amino acids.

  Ellagic acid used as an activator in the reagent of the present invention acts to activate a contact factor system (prekallikrein, high molecular weight kininogen, factor XII and factor XI) involved in the intrinsic coagulation pathway in the art. It is known to have

In the embodiment of the present invention, ellagic acid is not particularly limited, and naturally occurring or synthesized ellagic acid or a salt thereof can be used. Examples of ellagic acid salts include alkali metal salts of ellagic acid.
Ellagic acid exhibits a strong activation action by forming a chelate with a metal ion. Therefore, in the reagent of the present invention, ellagic acid is preferably in a state of forming a chelate with a metal ion such as Zn ²⁺ , Mn ²⁺ , or Al ³⁺ .

  In the present specification, the “amino acid having an aromatic ring” means an amino acid having a ring belonging to an aromatic group in the side chain. Examples of such an aromatic ring include a benzene ring, a condensed benzene ring, a non-benzene aromatic ring, and a heteroaromatic ring. The number of aromatic rings in the amino acid may be one or plural. When the amino acid has a plurality of aromatic rings, these aromatic rings may be the same or different from each other.

  In a preferred embodiment of the present invention, the amino acid having an aromatic ring is an α-amino acid. More preferably, the amino acid having an aromatic ring is at least one selected from the types of amino acids found in proteins and derivatives thereof. Such amino acids include phenylalanine, tyrosine, tryptophan, histidine, and derivatives thereof. The above-described derivative is a compound in which a hydrogen atom or a hydroxyl group in an aromatic ring contained in the above-described amino acid is arbitrarily substituted with an appropriate substituent. Such substituents are not particularly limited as long as they do not interfere with blood coagulation reaction and solubilization or dispersion of ellagic acid. Among the amino acids described above, phenylalanine and tyrosine are preferable as the amino acid having an aromatic ring, and phenylalanine is particularly preferable. The amino acid having an aromatic ring may be any of L-form, D-form and a mixture thereof. The amino acid having an aromatic ring may be a naturally occurring amino acid or a synthetic amino acid.

  In conventional clotting time measuring reagents, phenol is added as an ellagic acid precipitation inhibitor. In the reagent of the present invention, since the precipitation of ellagic acid can be prevented by the amino acid having the aromatic ring, phenol is not necessary for the reagent. That is, in the embodiment of the present invention, the coagulation time measurement reagent contains ellagic acid as an activator and an amino acid having an aromatic ring as its precipitation inhibitor, and does not contain phenol.

  The reagent of the present invention preferably further contains a phospholipid. Such phospholipids can be appropriately selected from phospholipids commonly used in clinical tests for blood coagulation ability. For example, phosphatidylethanolamine (hereinafter also referred to as “PE”), phosphatidylcholine (hereinafter also referred to as “PC”). And phosphatidylserine (hereinafter also referred to as “PS”). In the embodiment of the present invention, the coagulation time measurement reagent preferably contains at least one selected from PE, PC and PS. The phospholipid may be a naturally derived phospholipid or a synthetic phospholipid. Examples of naturally occurring phospholipids include phospholipids derived from rabbit brain, bovine brain, human placenta, soybean, egg yolk and the like. Among these, rabbit brain-derived phospholipid and soybean-derived phospholipid are preferable. In addition, the fatty acid side chain of phospholipid is not specifically limited, For example, a palmitic acid, an oleic acid, a stearic acid etc. are mentioned. Among them, oleic acid is particularly preferable.

  The reagent of the present invention may further contain a known activator other than ellagic acid. Such an activator is not particularly limited as long as it is a substance that can activate the contact factor system, and examples thereof include kaolin, celite, colloidal silica, and silicic anhydride.

  The aqueous medium used in the reagent of the present invention can be appropriately selected from aqueous media usually used for clinical examination of blood coagulation ability, and examples thereof include water and physiological saline. Moreover, as pH of the reagent of this invention, 6-8 are preferable and 7-7.6 are more preferable. In the embodiment of the present invention, the pH of the reagent can be adjusted by adding a buffer that is usually used for a reagent for measuring the clotting time. As such a buffering agent, a buffering agent having a buffering action at pH 5 to 9, preferably pH 6 to 8, can be suitably used, and examples thereof include HEPES, Tris, and MOPS.

  The reagent of the present invention may further contain an additive for improving its storage stability and stability. Such an additive may be any additive that is usually used for a reagent for measuring a coagulation time, and examples thereof include a preservative, an antioxidant, and a stabilizer. Examples of the preservative include sodium azide and known antibiotics. Examples of the antioxidant include butylhydroxyanisole. Examples of the stabilizer include polyethylene glycol and polyvinyl pyrrolidone.

  Since the reagent of the present invention contains ellagic acid as an activator, it can be suitably used for the measurement of APTT. In addition, the reagent of the present invention can also be used as a reagent for LA detection. In addition, the usage method of the reagent of this invention is the same as that of the conventional reagent for coagulation time measurement.

  The reagent of the present invention can be produced in the same manner as the conventional method for producing a coagulation time measurement reagent, except that an amino acid having an aromatic ring is used in place of phenol as a precipitation inhibitor for ellagic acid. An example of the method for producing the reagent of the present invention is as follows. First, ellagic acid is dissolved in an aqueous medium containing an appropriate base to prepare an aqueous solution of ellagic acid, and an amino acid having an aromatic ring is added to the aqueous solution. To do. And the reagent of this invention can be obtained by adjusting pH of the obtained mixture. If necessary, the above-described phospholipids, additives and the like may be further added to the obtained reagent.

  In an embodiment of the present invention, when the reagent is properly stored at a temperature of 1 to 30 ° C., ellagic acid does not precipitate for at least one month from the date of manufacture of the reagent. Here, “the precipitation of ellagic acid does not occur” means that the precipitation of ellagic acid is not visually confirmed.

  In the embodiment of the present invention, when a reagent is appropriately used together with a calcium salt solution under a general measurement condition of a coagulation time, a measurement result comparable to that of a conventional reagent for coagulation time measurement can be obtained. . More specifically, when a predetermined normal plasma (for example, Coagtrol IX manufactured by Sysmex Corporation) is measured as a sample using the reagent for measuring the clotting time of the present invention, the clotting time is in the range of 25 to 35 seconds. It is desirable that In addition, when a predetermined abnormal plasma (for example, Coagtrol IIX manufactured by Sysmex Corporation) is measured as a sample, it is desirable that the coagulation time is in the range of 60 to 100 seconds.

[2] Coagulation time measurement reagent kit The coagulation time measurement reagent kit of the present invention (hereinafter also simply referred to as “reagent kit”) includes ellagic acid as an activator and ellagic acid precipitation inhibitor, A first reagent containing an amino acid having an aromatic ring and a second reagent containing a calcium salt are included.

  Similar to the reagent of the present invention, the reagent kit of the present invention can be suitably used for APTT measurement and LA detection. The method of using the reagent kit of the present invention is the same as that of the conventional reagent kit for measuring the coagulation time.

  In the embodiment of the present invention, the reagent of the present invention can be suitably used as the first reagent. Therefore, the amino acid having an aromatic ring and ellagic acid contained in the first reagent are as described above. Moreover, it is preferable that a 1st reagent further contains said phospholipid. In the embodiment of the present invention, the first reagent may further contain at least one selected from the above-mentioned buffer, activator and additive.

  The concentration of ellagic acid in the first reagent can be appropriately set by those skilled in the art according to the measurement conditions of the coagulation time, but is usually 0.01 to 0.5 mM, preferably 0.05 to 0.2 mM. The concentration of the amino acid having an aromatic ring in the first reagent can be appropriately set by those skilled in the art depending on the type of the amino acid and the concentration of ellagic acid contained in the first reagent. Usually, 0.001 to 10 w / v% And 0.01 to 1.0 w / v% is preferable.

  Furthermore, the concentration of the phospholipid in the first reagent can be appropriately set by those skilled in the art depending on the type of phospholipid and the measurement conditions of the coagulation time, but is usually 30 to 2000 μg / mL, and is 100 to 600 μg / mL. preferable. In this case, when the phospholipid is PE, PC and PS, the PE concentration in the first reagent is usually 10 to 700 μg / mL, preferably 30 to 300 μg / mL. The PC concentration is usually 20 to 1000 μg / mL, preferably 30 to 500 μg / mL. The PS concentration is usually 3 to 300 μg / mL, preferably 5 to 150 μg / mL.

  The calcium salt contained in the second reagent may be any calcium salt usually used for clinical examination of blood coagulation ability, and can be selected from salts of calcium and inorganic acid or organic acid. Examples of such calcium salts include calcium chloride, calcium sulfate, calcium nitrite, calcium carbonate, calcium lactate, and calcium tartrate, among which calcium chloride is particularly preferable. The concentration of the calcium salt contained in the second reagent can be appropriately set by those skilled in the art depending on the type of calcium salt and the measurement conditions of the coagulation time, but is usually 15 to 50 mM, preferably 20 to 30 mM. . When calcium chloride is used as the calcium salt, the concentration is preferably 15 to 30 mM, and more preferably 20 to 25 mM.

  The reagent kit of the present invention may further contain a reagent or the like usually used for measurement of the coagulation time as another reagent. Examples of such a reagent include an aqueous medium for dilution and plasma for reference. Examples of the aqueous medium for dilution include physiological saline and buffer for diluting the first reagent. For example, when detecting LA with the reagent kit of the present invention, the first reagent containing phospholipid is diluted with such an aqueous medium for dilution to prepare two kinds of first reagents having different phospholipid concentrations. Are preferably used.

  Examples of the reference plasma include normal plasma and accuracy control plasma. When LA is detected with the reagent kit of the present invention, it is preferable to further include plasma lacking various coagulation factors and LA positive plasma as reference plasma.

[3] Coagulation time measurement method The coagulation time measurement method of the present invention (hereinafter also simply referred to as “measurement method”) includes a blood sample, ellagic acid as an activator, and an ellagic acid precipitation inhibitor, A first mixing step of mixing a first reagent containing an amino acid having an aromatic ring;
A second mixing step of mixing the sample obtained in the first mixing step with a second reagent containing a calcium salt;
And a step of measuring the coagulation time of the sample obtained in the second mixing step.

  As the blood sample, blood or plasma obtained from the blood is used. In a preferred embodiment of the present invention, plasma obtained from blood collected from a subject (hereinafter also referred to as “test plasma”), the above-mentioned reference plasma, and a mixture thereof are used as blood samples. A method for obtaining plasma from blood is known in the art. For example, plasma can be obtained by centrifuging so as not to hemolyze blood and removing blood cell components. In addition, blood collected from a subject may be added with a known anticoagulant usually used for clinical examination of blood coagulation ability. Examples of such an anticoagulant include sodium citrate.

  For the purpose of detecting LA, it is preferable to further use a mixture of test plasma and normal plasma in which LA may be present as a blood sample. When the subject is deficient in clotting factors, such mixing with normal plasma prevents the prolongation of the clotting time and improves the sensitivity of the test. The mixing ratio (volume ratio) of test plasma and normal plasma is usually 8: 2 to 2: 8, preferably 5: 5.

  In the first mixing step, the blood sample and the first reagent are mixed. Here, as the first reagent, the above-described reagent of the present invention or the first reagent of the reagent kit can be suitably used. For the purpose of detecting LA, it is preferable to dilute the first reagent containing phospholipid to prepare and use two kinds of first reagents having different phospholipid concentrations.

  The mixing ratio (volume ratio) of the blood sample and the first reagent in the first mixing step is usually 8: 2 to 2: 8, preferably 6: 4 to 4: 6, and more preferably 5: 5. It is. In the first mixing step, it is preferable to perform incubation. The incubation time is usually 1 to 10 minutes, preferably 3 to 5 minutes. The temperature condition is usually 25 to 45 ° C, preferably 35 to 38 ° C.

  In the second mixing step, the sample obtained in the first mixing step and the second reagent are mixed. Here, as the second reagent, the second reagent of the above-described reagent kit of the present invention can be suitably used. The mixing ratio (volume ratio) of the sample and the second reagent is usually 8: 2 to 5: 5, preferably 7: 3 to 6: 4, and more preferably 2: 1. Moreover, temperature conditions are the same as that of a 1st mixing process.

  In the step of measuring the coagulation time, the coagulation time of the sample obtained in the second mixing step is measured. The coagulation time measuring device is not particularly limited as long as it is a known device used in general APTT measurement. For example, the coagulation time can be measured by a device that measures optical information obtained from the sample. Such optical information includes changes in transmitted light, changes in scattered light, and the like. Moreover, as a measuring apparatus, the commercially available blood coagulation measuring apparatus provided with an optical information detection part can be used conveniently, for example, Sysmex Corporation CS-2000i and CS-2100i are mentioned.

  For the purpose of detecting LA, it is preferable to use a first reagent containing phospholipid, a reagent diluted with the first reagent, and a second reagent for each of test plasma and normal plasma. In addition, the detection of LA may be performed based on the comparison result by comparing a value obtained based on the clotting time obtained from the test plasma with the above-described two kinds of first reagents and a threshold value described later. preferable.

The above threshold is more preferably determined based on the ratio of the clotting time of the test plasma to the clotting time of normal plasma, such as the Lupus ratio (hereinafter also referred to as “LR value”) and the Rosner index. .
Here, the LR value is a value calculated by the following formula (I).
Formula (I): LR value = (b / a) / (d / c) = bc / ad
(In formula (I), a, b, c and d represent the following measured values, respectively: a: APTT obtained using test plasma and first reagent, b: first reagent diluted with test plasma. C: APTT obtained using normal plasma and first reagent, d: APTT obtained using normal plasma and diluted first reagent)

  In the case of normal plasma, the LR value is about 1. In the case of plasma obtained from a blood coagulation factor-deficient patient, a warfarin-administered patient, or a heparin-administered patient, although the clotting time is prolonged as compared with normal plasma, the LR value is about 1 as a result. In contrast, LR values are greater than 1 for plasma obtained from LA positive patients. Therefore, LA in the test plasma can be detected from the comparison result of the LR values between the test plasma and the normal plasma.

  EXAMPLES The present invention will be described in detail below by examples, but the present invention is not limited to these examples.

Example 1: Examination of effect of preventing precipitation of ellagic acid by addition of amino acid having aromatic ring (1-1) Preparation of reagent for measuring coagulation time
Various coagulation time measurement reagents based on the APTT measurement principle were prepared as follows. Hereinafter, “%” indicates “w / v%”.
<Reagent A>
As a reagent A, a reagent containing ellagic acid as an activator but not containing an amino acid having a phenol and an aromatic ring was prepared. Reagent A is 50 mM HEPES (molecular weight: 238.30, Dojindo Laboratories), 25 mM Tris (molecular weight: 121.14, Nacalai Tesque), 1% glycine (molecular weight: 75.07, Nacalai Tesque), 0.05% Polyvinylpyrrolidone K-30 (molecular weight: about 40,000, Kishida Chemical Co., Ltd.) and 1% polyethylene glycol 6000 (molecular weight: about 9,000, Nacalai Tesque Co., Ltd.) in a solution of 0.1 mM ellagic acid (molecular weight: molecular weight: 338.22, Tokyo Chemical Industry Co., Ltd.) and 140 μg / mL phosphatidylethanolamine (molecular weight: 744.04, Avanti Polar Lipids, Inc.), 160 μg / mL phosphatidylcholine (molecular weight: 786.15, Avanti Polar Lipids, Inc.) and 20 μg as phospholipids / mL phosphatidylserine (molecular weight: 810.03, Avanti Polar Lipids, Inc.) and 3-t-butyl-4-hydroxyanisole (molecular weight: 1) as an antioxidant 80.25, Nacalai Tesque Co., Ltd.).

<Reagent B>
As reagent B, a conventional reagent containing ellagic acid and phenol as its precipitation inhibitor was prepared. Reagent B is a reagent containing 0.35% phenol (molecular weight: 94.11, Kishida Chemical Co., Ltd.) in the above-mentioned reagent A.

<Reagent C>
As a reagent C, a reagent containing ellagic acid and an amino acid having an aromatic ring (phenylalanine) but not containing phenol was prepared. Reagent C is a reagent containing 0.1% phenylalanine (molecular weight: 165.19, SIGMA-ALDRICH) in the above-mentioned reagent A.
<Reagent D>
As a reagent D, a reagent containing ellagic acid and an amino acid having an aromatic ring (tyrosine) but not containing phenol was prepared. Reagent D is a reagent in which 0.01% tyrosine (molecular weight: 181.19, Kishida Chemical Co., Ltd.) is added to reagent A described above.

Note that the pH of each of the reagents A to D was adjusted to 7.4. Further, ZnCl ₂ and AlCl ₃ are added to these reagents, and ellagic acid in a state of forming a chelate with a metal ion is included.

(1-2) Confirmation of ellagic acid precipitation After the reagents A to D prepared in (1) above were allowed to stand at 4 ° C. or room temperature for 1 month, the presence or absence of ellagic acid precipitation in each reagent was visually confirmed. did. The results are shown in Table 1 below.

  From Table 1, precipitation was caused by aggregation of ellagic acid in Reagent A containing neither phenol nor an amino acid having an aromatic ring, but precipitation was confirmed in Reagent B containing phenol as in the conventional product. There wasn't. In addition, it was confirmed that precipitation did not occur even in Reagent C and Reagent D containing an amino acid having an aromatic ring. From this, it was shown that precipitation of ellagic acid can be prevented by adding an amino acid having an aromatic ring to the reagent as an ellagic acid precipitation inhibitor instead of phenol.

Example 2: Examination of influence on APTT measurement ability by addition of amino acid having aromatic ring (2-1) Test plasma Coagtrol IX (Sysmex Corporation) as normal plasma and Coagtrol IIX (Sysmex Corporation) as abnormal plasma Using.

(2-2) First Reagent and Second Reagent Reagents A to D prepared in Example 1 were used as the first reagent. A 25 mM calcium chloride solution was used as the second reagent.

(2-3) Measurement of coagulation time The above two types of plasma (each 50 μL) were heated at 37 ° C. for 1 minute, then mixed with the first reagent (50 μL), and further heated for 3 minutes. Thereafter, the mixture of plasma and the first reagent and the second reagent (50 μL) were mixed, and the clotting time was measured. The measurement of the coagulation time was performed using a fully automatic blood coagulation analyzer Coagrex 800 (manufactured by Sysmex Corporation). Measurement was performed twice for both normal plasma and abnormal plasma. The measurement results (average values) are shown in Table 2 below.

  From Table 2, the clotting time with Reagent C and Reagent D containing an amino acid having an aromatic ring as an ellagic acid precipitation inhibitor was within the specifications for both normal and abnormal plasma samples. These results were comparable to the coagulation time with reagent B containing phenol. Therefore, it was shown that the reagent of the present invention has the same level of measuring ability as that of the conventional clotting time reagent.

Claims (9)

  A reagent for measuring a coagulation time, comprising ellagic acid as an activator and an amino acid having an aromatic ring.   The coagulation time measurement reagent according to claim 1, wherein the amino acid having an aromatic ring is at least one selected from the types of amino acids found in proteins and derivatives thereof.   The reagent for coagulation time measurement according to claim 1 or 2, wherein the amino acid having an aromatic ring is at least one selected from phenylalanine and tyrosine.   The coagulation time measurement reagent according to any one of claims 1 to 3, further comprising a phospholipid.   The reagent for measuring a coagulation time according to claim 4, wherein the phospholipid is at least one selected from the group consisting of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine.   The reagent for coagulation time measurement according to any one of claims 1 to 5, which is a reagent for measuring activated partial thromboplastin time or detecting lupus anticoagulant.   A reagent kit for measuring a coagulation time, comprising: a first reagent containing ellagic acid as an activator, and an amino acid having an aromatic ring; and a second reagent containing a calcium salt.   The reagent kit for coagulation time measurement according to claim 7, wherein an amino acid having an aromatic ring is contained in the first reagent in an amount of 0.01 to 1.0 w / v%. A first mixing step of mixing a blood sample with ellagic acid as an activator and a first reagent containing an amino acid having an aromatic ring;
A second mixing step of mixing the sample obtained in the first mixing step with a second reagent containing a calcium salt;
A coagulation time measurement method including a step of measuring the coagulation time of the sample obtained in the second mixing step.