JP2005249778A - Reagent kit for detecting lupus anticoagulant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent kit capable of specifically and highly precisely detecting a lupus anticoagulant by heightening detection sensitivity to a lupus anticoagulant and discriminating it from a specimen containing a blood coagulation inhibitor such as a coagulation factor deficiency and heparin. <P>SOLUTION: In the lupus anticoagulant detection reagent kit, the content of phosphatidyl ethanol amine is adjusted in reagents having different contents of phosphatidyl serine to phospholipid contained in the reagents. It is thereby possible to heighten detection sensitivity to a lupus anticoagulant. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a blood coagulation reagent kit used for diagnosis of antiphospholipid antibody syndrome in fields such as clinical chemistry and pharmaceutical research. More specifically, in vitro blood tests specifically detect lupus anticoagulant-positive diseases. The present invention relates to a reagent kit that can be used.

  Lupus anticoagulant (hereinafter abbreviated as LA) is the first reported anticoagulant for SLE (Systemic Lupus Erythematosus) patients. While the frequency of detection of LA in SLE patients is 5% to 10%, it may be detected in other autoimmune diseases and neoplastic diseases. LA is frequently detected in antiphospholipid syndrome (APS) such as thrombosis, premature labor, thrombocytopenia, and the time until coagulation is prolonged due to a phospholipid-dependent coagulation reaction. It is believed to be an autoantibody against phospholipids.

  In recent years, it has been revealed that LA is a heterogeneous antibody and is an antibody against a complex of negatively charged phospholipid and β2-glycoprotein I (β2-GPI) or prothrombin. The anticoagulant action of LA is thought to occur because LA binds to this complex to inhibit the change of prothrombin to thrombin.

  As a reagent for detecting LA having such characteristics, a coagulation reagent containing a certain amount of phospholipid, for example, rabbit brain-derived cephalin, bovine brain-derived cephalin, soybean lecithin, snake venom and the like has been conventionally used. If LA is present in the sample blood, LA will neutralize the phospholipids in the reagent, so the phospholipids necessary for the start of the cascade reaction to coagulate the sample will be insufficient. In proportion to the amount of phospholipid produced, the clotting time of each test is prolonged, and this can be diagnosed as LA positive.

  In addition, since the detection sensitivity of LA increases as the phospholipid in the coagulation reagent decreases, two or more different methods are combined to dilute the reagent or to ensure accuracy. For example, using an activated partial thromboplastin time measurement (hereinafter abbreviated as APTT) reagent in which two reagents having different phospholipid concentrations are combined, the coagulation time of each reagent is used to determine the Rossner Index and the Lupus ratio. A method is known in which (Lupus Ratio) (LR value) is calculated and the LA positive rate is determined.

  As a commercially available coagulation measuring reagent, there is a test reagent based on the Russell snake venom method called Gradipore LA (Gradipore, Australia). This is a reagent kit in which a first reagent containing snake venom and a second reagent containing snake venom and excess soybean phospholipid are combined, and the coagulation time when the first reagent is added / coagulation time when the second reagent is added. When the ratio obtained in (1) is 1.3 or more, it is determined as LA positive.

  Furthermore, according to existing research reports, LA is arithmetically detected using the difference in phospholipid concentration due to rabbit brain-derived phospholipids and the like (Non-patent Document 1).

  Furthermore, when a sample in which normal plasma and patient plasma are mixed at a ratio of 1: 1 is used, an APTT reagent or a diluted Russell snake venom measurement (dRVVT) reagent in which two types of reagents having different phospholipid concentrations are combined is used. A method is also used in which LA is detected using the degree of clotting time of a patient's plasma as an index.

V. Chantarangkul, et al. , Thromb. Res. 1992, 67: 355-365; Rossner, et al. , Thromb. Haemost. 1987, 57: 144-147

  In the conventional phospholipid-dependent coagulation measurement, naturally derived phospholipids were used, and the phospholipid composition was not adjusted. Since naturally derived phospholipids are heterogeneous, this affects the clotting time, and there is a problem that the detection sensitivity of LA is considerably different. In addition, the coagulation test can normalize the coagulation time by mixing samples other than LA with normal plasma to replenish deficient coagulation factors, but blood such as warfarin and heparin It is difficult to distinguish between a sample containing a coagulation inhibitor and LA positive patient plasma, and there is a problem that these samples containing a coagulation inhibitor can also be determined as LA positive.

  Therefore, in order to accurately determine the LA disease, it is necessary to distinguish between an increase in the coagulation time due to a coagulation activity inhibitor other than LA or a coagulation factor deficiency, and an increase in the coagulation time due to LA.

  An object of the present invention is to provide a reagent kit capable of distinguishing from heparin-administered patient plasma and warfarin-administered patient plasma, enhancing LA detection sensitivity, and specifically detecting even a weak LA positive sample.

  As a result of intensive studies to solve the above problems, the present inventor has determined that the plasma of heparin-administered patients by adjusting the content of phosphatidylethanolamine in a reagent having a different content of phosphatidylserine relative to the phospholipid contained in the reagent. It is intended to provide a reagent kit for detection of LA that can be specifically detected even if it is a weak LA positive sample, while distinguishing it from patient plasma administered with warfarin and increasing LA detection sensitivity.

A first aspect of the present invention includes a first coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine, and a second coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine. ,
The reagent kit for detecting lupus anticoagulant, wherein the concentration of phosphatidylethanolamine of the first clotting time measurement reagent is 40 to 280 μM and the concentration of phosphatidylethanolamine of the second clotting time measurement reagent is 1 to 30 μM About.

The second aspect of the present invention comprises a first coagulation time measurement reagent comprising a first partial reagent and a second partial reagent, and a second coagulation time measurement reagent comprising a third partial reagent and a fourth partial reagent,
The first partial reagent contains a phospholipid containing phosphatidylserine and phosphatidylethanolamine, the third partial reagent contains a phospholipid containing phosphatidylserine and phosphatidylethanolamine,
The present invention relates to a reagent kit for detecting lupus anticoagulant, wherein the first partial reagent has a phosphatidylethanolamine concentration of 40 to 280 μM and the third partial reagent has a phosphatidylethanolamine concentration of 1 to 30 μM.

  In the reagent kit of the present invention, heparin-administered patient plasma and warfarin-administered patient plasma are distinguished from each other, and LA detection sensitivity is enhanced so that even a weak LA positive sample can be specifically detected.

  The LA detection reagent kit includes a first coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine, and a second coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine, The concentration of phosphatidylethanolamine contained in the first clotting time measurement reagent is 40 to 280 μM, and the concentration of phosphatidylethanolamine contained in the second clotting time measurement reagent is 1 to 30 μM.

  Furthermore, the ratio of the phosphatidylserine content contained in the first clotting time measurement reagent to the total phospholipid content is different from the ratio of the phosphatidylserine content contained in the second clotting time measurement reagent to the total phospholipid content. Is preferred.

  The phosphatidylethanolamine (hereinafter sometimes abbreviated as PE) and phosphatidylserine (hereinafter sometimes abbreviated as PS) contained in the first clotting time measuring reagent and the second clotting time measuring reagent are naturally derived. However, since naturally derived phospholipids are heterogeneous, this affects the clotting time, and the detection sensitivity of LA is considerably different. Moreover, when using a naturally derived thing, it is preferable to use what was refine | purified to 99% or more of purity from said point.

Examples of phospholipids other than PE and PS contained in the first coagulation time measurement reagent and the second coagulation time measurement reagent include phosphatidylcholine (hereinafter sometimes abbreviated as PC) as phospholipids that promote blood coagulation. Is mentioned. PC may be naturally derived as well, but is preferably a synthetic product. Similarly, when using a naturally-derived material, it is preferable to use a material purified to a purity of 99% or more.
Various fatty acid side chains of phospholipids such as PE, PS, and PC are used, preferably palmitic acid, oleic acid, stearic acid, and more preferably oleic acid.

  The first coagulation time measurement reagent and the second coagulation time measurement reagent may further contain other components necessary for causing blood coagulation in vitro. Examples of other components include an activator, snake venom, calcium, and tissue factor. As the activator, it is preferable to use one or more selected from the group consisting of ellagic acid, kaolin, celite, and colloidal silica. As the snake venom, it is preferable to use one or more selected from the group consisting of Russell snake venom, textural snake venom and ecarin snake venom. As tissue factor, those derived from rabbit brain or human placenta or recombinant are preferably used.

  These other components are appropriately selected according to the type of coagulation time measurement reagent. For example, when the first coagulation time measurement reagent and the second coagulation time measurement reagent contain calcium and an activator, the coagulation time is measured based on the measurement principle of activated partial thromboplastin time measurement. When the first coagulation time measurement reagent and the second coagulation time measurement reagent contain calcium and snake venom, the coagulation time is measured based on the principle of Russell snake venom measurement. When the first clotting time measuring reagent and the second clotting time measuring reagent contain calcium and tissue factor, the clotting time is measured based on the principle of prothrombin time measurement.

  The first clotting time measuring reagent and the second clotting time measuring reagent may further contain a buffer such as Hepes and Tris as necessary. The concentration of the buffer solution may be a concentration generally used in the field of clinical chemistry, and can be determined by simple repeated experiments.

The phospholipid content of the first clotting time measuring reagent and the second clotting time measuring reagent is obtained by mixing an equal amount of the sample and the first clotting time measuring reagent or the second clotting time measuring reagent (50 μL of the sample and the first clotting time The measurement method for measuring the coagulation time (when mixing 50 μL of the measurement reagent or the second coagulation time measurement reagent) is as follows. The PE concentration in the first coagulation time measurement reagent is 40 to 280 μM, preferably 65 to 269 μM, more preferably 100 to 200 μM, and the PS concentration is 30 to 370 μM, preferably 37 to 250 μM, more preferably 60 to 250 μM. Yes, the PC concentration is 10 to 140 μM, more preferably 25 to 64 μM.
The PE concentration in the second clotting time measurement reagent is 1 to 30 μM, preferably 1 to 27 μM, more preferably 6 to 21 μM, the PS concentration is 1 to 25 μM, more preferably 6 to 19 μM, and the PC concentration Is 10 to 140 μM, more preferably 25 to 64 μM. A low PE concentration is not preferable because the coagulation time is extended.

  The concentration of the above-mentioned phospholipid is the concentration when 50 μL of the sample is mixed with 50 μL of the first clotting time measuring reagent or the second clotting time measuring reagent, but when the mixing ratio of the sample and the clotting time measuring reagent is different, The final concentration of phospholipid after mixing the first clotting time measuring reagent and the second clotting time measuring reagent may be in the same range as in the above case.

  Moreover, it is preferable that the PE concentration or the PS concentration contained in the first coagulation time measurement reagent is 3 to 15 times the PE or PS concentration contained in the second coagulation time measurement reagent. If the magnification is low, it is difficult to distinguish LA positive patients from heparin-administered patient plasma and warfarin-administered patient plasma. Further, if the magnification is high, the difference in the clotting time of normal plasma becomes large between the first clotting time measuring reagent and the second clotting time measuring reagent, which is not preferable.

  Further, the first clotting time measuring reagent and the second clotting time measuring reagent may be a mixture of phospholipid and the above-mentioned other components, respectively.

The first clotting time measurement reagent is composed of a first partial reagent and a second partial reagent accommodated in separate containers, and the second partial clotting time measurement reagent is accommodated in separate containers and the fourth partial reagent. You may be comprised from a partial reagent. The PE concentration in the first partial reagent is 40 to 280 μM, preferably 65 to 269 μM, more preferably 100 to 200 μM, and the PS concentration is 30 to 370 μM, preferably 37 to 250 μM, more preferably 60 to 250 μM. The PC concentration is 10 to 140 μM, more preferably 25 to 64 μM.
The PE concentration of the third partial reagent is 1 to 30 μM, preferably 1 to 27 μM, more preferably 6 to 21 μM, the PS concentration is 1 to 25 μM, more preferably 6 to 19 μM, and the PC concentration is 10 to 10 μM. 140 μM, more preferably 25 to 64 μM.

  In the coagulation time measurement reagent based on the principle of activated partial thromboplastin time measurement, the first coagulation time measurement reagent is composed of the first partial reagent and the second partial reagent, and the second coagulation time measurement reagent is the third partial reagent and the fourth part. Examples include a reagent, a first partial reagent and a third partial reagent phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and an activating agent, and the second partial reagent and the fourth partial reagent containing calcium.

  In the coagulation time measurement reagent based on the principle of Russell snake venom measurement, the first coagulation time measurement reagent is composed of the first partial reagent and the second partial reagent, and the second coagulation time measurement reagent is composed of the third partial reagent and the fourth partial reagent. In addition, the first partial reagent and the third partial reagent phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and snake venom are contained, and the second partial reagent and the fourth partial reagent contain calcium.

  In the coagulation time measurement reagent based on the principle of prothrombin time measurement, the first coagulation time measurement reagent is composed of the first partial reagent and the second partial reagent, and the second coagulation time measurement reagent is composed of the third partial reagent and the fourth partial reagent. In addition, the first partial reagent and the third partial reagent phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and tissue factor are contained, and the second partial reagent and the fourth partial reagent contain calcium.

  The reagent kit of the present invention includes a first coagulation time measurement reagent comprising the first partial measurement reagent and the second partial measurement reagent having the above composition, and a second partial measurement reagent comprising the third partial measurement reagent and the fourth partial measurement reagent. This is a combination of two types of reagents with different coagulation times in the coagulation time measurement reagent.

Next, a method for detecting LA using the reagent kit of the present invention having the above configuration will be described.
First, the specimen sample is divided into two, the first clotting time measuring reagent is added to one, and the second clotting time measuring reagent is added to the other specimen sample. When the coagulation time measurement reagent is divided into the first partial reagent and the second partial reagent, respectively, the first partial reagent and the second partial reagent may be mixed and then added to the specimen. After adding the first partial reagent (or second partial reagent) to the specimen, the second partial reagent (or first partial reagent) may be added. When adding the first partial reagent or the second partial reagent separately, after adding the first partial reagent (or the second partial reagent), before adding the second partial reagent (or the first partial reagent), if necessary, the specimen And the mixture of partial reagents may be heated.
After each reagent is added to the specimen, the clotting time of the sample is measured. The coagulation time may be measured by a known method, for example, using a known automatic measuring device.

  Here, as the sample used for the specimen, it is preferable to use plasma rather than using blood as it is, and more preferably platelet-free plasma. More preferably, the patient's plasma is mixed with normal plasma or platelet-depleted normal plasma. By mixing normal plasma, it is possible to prevent the prolongation of the coagulation time based on the coagulation factor deficiency, and the sensitivity of the phospholipid-dependent blood coagulation test is increased. The mixing ratio of patient plasma and normal plasma is generally 4: 1 to 1: 4, preferably 1: 1.

  In the coagulation test using the reagent kit of the present invention, the coagulation time of samples derived from anticoagulant diseases such as LA positive patients, blood coagulation factor deficient patients, warfarin administration patients, heparin administration patients, etc. become longer. Further, for LA positive patient specimens, a difference in coagulation time between the first coagulation time measurement reagent and the second coagulation time measurement reagent is also observed. That is, in the sample derived from an LA positive patient, the coagulation time when the second coagulation time measurement reagent is used is longer than the coagulation time when the first coagulation time measurement reagent is used. On the other hand, in the samples derived from blood coagulation factor-deficient patients, warfarin-administered patients, and heparin-administered patients, no significant difference in coagulation time was observed between the first coagulation time measurement reagent and the second coagulation time measurement reagent. Therefore, LA can be specifically detected based on the difference in coagulation time between the first coagulation time measurement reagent and the second coagulation time measurement reagent.

  The detection of LA can be determined by the difference in the clotting time measured with the first clotting time measuring reagent and the second clotting time measuring reagent. The ratio of the clotting time of the sample from, for example, Rossner Index (E. Rossner, et al., Thromb. Haemost. 1987, 57: 144-147) or Lupus Ratio (LR value) (R. Schjetlein, et al., Thromb. Res. 1993, 69: 239-250).

In the case of normal plasma, the LR value calculated using the first clotting time measuring reagent and the second clotting time measuring reagent is about 1. In addition, the clotting time is prolonged as compared with normal plasma even in cases derived from a blood coagulation factor deficient patient, a warfarin-administered patient, or a heparin-administered patient, but the clotting of the first clotting time reagent and the second clotting time reagent. Since almost no time difference is observed, the LR value is about 1. On the other hand, in the case of LA positive, the second coagulation time measurement reagent measures the first coagulation time, although it depends on the concentration and combination of the first coagulation time measurement reagent and the second coagulation time measurement reagent. Since the coagulation time is longer than that of the reagent, the specimen from the LA positive patient can be automatically detected by comparing the LR values.

(Example 1)
Examination of PE concentration Hepes, ellagic acid, Tris, phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were mixed to prepare the first partial reagents 1-6 and the third partial reagent.
The concentration of each component of the first partial reagent 1 was 50 mM Hepes, 0.1 mM ellagic acid, 25 mM Tris, 123 μM PS, 38 μM PC, and 13 μM PE. Further, first partial reagents 2 to 6 were prepared in the same manner as the first partial reagent 1 except that the PE concentration was changed to 67 μM, 101 μM, 134 μM, 202 μM, and 269 μM. The pH of the first partial reagents 1-6 was 7.35.
The concentration of each component of the third partial reagent was 50 mM Hepes, 0.1 mM ellagic acid, 25 mM Tris, 12 μM PS, 13 μM PE, and 38 μM PC. The pH was 7.35. PE, PS, and PC used synthetic phospholipids whose fatty acid side chains are oleic acid.

Moreover, what was prepared with purified water so that a calcium chloride concentration might be set to 25 mM was used as the 2nd partial reagent and the 4th partial reagent.
Nine types of strongly LA positive patient plasma, nine types of LA weakly positive patient plasma, and five types of warfarin-administered patient plasma were obtained. Each patient plasma was mixed with normal plasma at a ratio of 1: 1 as a measurement sample.

  Prepare two sets of normal plasma and 50 μL of each measurement sample prepared above, add 50 μL of the first partial reagent 1 (or third partial reagent) to each sample in one set, and warm to 37 ° C. for 3 minutes After that, 50 μL of the second partial reagent (or the fourth partial reagent) was added to each sample. The clotting time was measured using a fully automatic blood coagulation analyzer Coagrex 800 (Shimadzu Corporation). The coagulation time was measured in the same manner except that the first partial reagents 2 to 6 were used instead of the first partial reagent 1.

From the coagulation time measured for each sample, the Lupus Ratio (LR value) represented by the following formula 1 was calculated. Calculation of LR value was performed based on literature (R. Schjetlein, et al., Thromb. Res. 1993, 69: 239-250).
[Formula 1]
Lupus Ratio (LR) = (b / a) / (d / c) = bc / ad
Here, “a”, “b”, “c”, and “d” in Formula 1 represent measured values obtained by combinations shown in Table 1.

  The measurement results are shown in FIG. FIG. 1 is a bar graph showing the average of LR values when 9 types of LA strongly positive patient plasma, 9 types of LA weakly positive patient plasma and 5 types of warfarin-administered patient plasma are measured, and LR when each sample is measured. The standard deviation (SD) of the values is indicated by a bar. The horizontal axis indicates the PE concentration in the first partial reagents 1-6. As shown in FIG. 1, it has been clarified that the use of the first partial reagents 1 to 6 enables differentiation between warfarin-administered patient plasma and LA weakly positive patient plasma and LA strongly positive patient plasma.

(Example 2)
Examination of concentration of PS Hepes, ellagic acid, Tris, phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were mixed to prepare the first partial reagent 7-12 and the third partial reagent.
The concentration of each component of the first partial reagent 7 was 50 mM Hepes, 0.1 mM ellagic acid, 25 mM Tris, 67 μM PE, 38 μM PC, and 37 μM PS. Further, first partial reagents 8 to 12 were prepared in the same manner as the first partial reagent 7, except that the PS concentration was changed to 62 μM, 93 μM, 123 μM, 185 μM, and 247 μM. The pH of the first partial reagents 7-12 was 7.35.
The concentration of each component of the third partial reagent was 50 mM Hepes, 0.1 mM ellagic acid, 25 mM Tris, 12 μM PS, 13 μM PE, and 38 μM PC. The pH was 7.35. PE, PS, and PC used synthetic phospholipids whose fatty acid side chains are oleic acid.
Moreover, what was prepared with purified water so that a calcium chloride concentration might be set to 25 mM was used as the 2nd partial reagent and the 4th partial reagent. The coagulation time was measured in the same manner as in Example 1 except that each reagent prepared here was used.

  The measurement results are shown in FIG. FIG. 2 is a bar graph showing the average of LR values when 9 types of LA strongly positive patient plasma, 9 types of LA weakly positive patient plasma and 5 types of warfarin-administered patient plasma are measured, and LR when each sample is measured. The standard deviation (SD) of the values is indicated by a bar. The horizontal axis indicates the PE concentration in the first partial reagents 7-12. As shown in FIG. 2, it was revealed that the use of the first partial reagents 7 to 12 can distinguish the plasma of warfarin-administered patient from the weakly LA positive patient plasma and the strongly LA positive patient plasma. In addition, the difference in the LR value between warfarin-administered patient plasma, LA weakly positive patient plasma, and LA strongly positive patient plasma was increased until the PS concentration in the first partial reagent was 123 μM. Became almost constant.

(Example 3)
Examination of reactivity of various specimens LA strongly positive specimens (Sample Nos. 1 to 9 shown in Table 2), LA weakly positive specimens (Sample Nos. 10 to 17 shown in Table 2), Warfarin administered patient specimens (Sample Nos. Shown in Table 2) 18-22), VIII factor-deficient patient plasma samples (sample numbers 23 to 25 shown in Table 2), heparin-administered patient plasma samples (sample numbers 26 and 27 shown in Table 2) (reagents used in Example 1) Measurement was performed using a 1-part reagent 1-6, a second-part measurement reagent, a third-part measurement reagent, and a fourth-part measurement reagent) and a diluted Russell snake venom reagent (dRVVT shown in Table 2), which is a commercially available LA measurement reagent.

The operation was performed in the same manner as in Example 1, and LR was measured. The measurement of each specimen using the diluted Russell snake venom reagent was performed by measuring the coagulation time according to the dosage and calculating the “ratio of coagulation time”. In addition, those having “LR value” and “coagulation time ratio” of 1.2 or more were determined to be LA positive.
The results are shown in Table 2.

As shown in Table 2, when the diluted Russell snake venom reagent was used, specimen numbers 15 and 16 which were LA weak positive specimens were judged negative, and specimen numbers 20 and 21 which were warfarin-administered patient specimens and heparin-administered patient specimens. Sample No. 27 was determined to be positive.
In addition, when the first partial reagent 1 having a PE concentration of 13 μM was used, it was determined to be negative for sample number 15 which was a weak LA positive sample.
However, by using the first partial measurement reagents 2 to 6, it is determined that both the LA strong positive patient sample and the LA weak positive sample are positive, and for the warfarin administration patient sample, the factor VIII deficient patient plasma sample, and the heparin administration patient plasma sample A reagent kit for LA detection that is judged negative and distinguishes LA positive patient specimens from palin-administered patient plasma and warfarin-administered patient plasma, increases LA detection sensitivity, and can specifically detect even weak LA positive specimens It became clear that it could be provided.

  As described above, by adjusting the content of phosphatidylethanolamine in the reagent having a different content of phosphatidylserine relative to the phospholipid contained in the reagent, it is possible to distinguish heparin-administered patient plasma and warfarin-administered patient plasma, and It is possible to increase the detection sensitivity and supply an LA detection reagent kit that can specifically detect even a weak LA positive sample.

It is a figure which shows the graph of the relationship between PE density | concentration in a 1st partial reagent, and LR value. It is a figure which shows the graph of the relationship between PS density | concentration in a 1st partial reagent, and LR value.

Claims (16)

A first coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine, and a second coagulation time measurement reagent containing a phospholipid containing phosphatidylserine and phosphatidylethanolamine,
The reagent kit for detecting lupus anticoagulant, wherein the concentration of phosphatidylethanolamine of the first clotting time measurement reagent is 40 to 280 μM and the concentration of phosphatidylethanolamine of the second clotting time measurement reagent is 1 to 30 μM . The reagent kit for detecting lupus anticoagulant according to claim 1, wherein the concentration ratio of phosphatidylethanolamine or phosphatidylserine between the first clotting time measuring reagent and the second clotting time measuring reagent is 3-15. The reagent kit for detecting lupus anticoagulant according to claim 1 or 2, wherein the first coagulation time measurement reagent and the second coagulation time measurement reagent contain phosphatidylcholine. The reagent kit for detecting lupus anticoagulant according to any one of claims 1 to 3, wherein the first coagulation time measurement reagent and the second coagulation time measurement reagent contain an activator and calcium ions. The reagent kit for detecting lupus anticoagulant according to claim 4, wherein the activator is selected from the group consisting of ellagic acid, kaolin, celite, and colloidal silica. The reagent kit for detecting lupus anticoagulant according to any one of claims 1 to 3, wherein the first coagulation time measurement reagent and the second coagulation time measurement reagent contain snake venom and calcium ions. The reagent kit for detecting lupus anticoagulant according to any one of claims 1 to 3, wherein the first coagulation time measurement reagent and the second coagulation time measurement reagent contain a tissue factor and calcium ions. A first clotting time measuring reagent comprising a first partial reagent and a second partial reagent, and a second clotting time measuring reagent comprising a third partial reagent and a fourth partial reagent,
The first partial reagent contains a phospholipid containing phosphatidylserine and phosphatidylethanolamine, the third partial reagent contains a phospholipid containing phosphatidylserine and phosphatidylethanolamine,
A reagent kit for detecting lupus anticoagulant, wherein the concentration of phosphatidylethanolamine in the first partial reagent is 40 to 280 μM and the concentration of phosphatidylethanolamine in the third partial reagent is 1 to 30 μM. The reagent kit for detecting lupus anticoagulant according to claim 8, wherein the concentration ratio of phosphatidylethanolamine or phosphatidylserine to the first partial reagent and the third partial reagent is 3 to 15. The reagent kit for detecting lupus anticoagulant according to claim 8 or 9, wherein the first partial reagent and the third partial reagent contain phosphatidylcholine. The reagent kit for detecting lupus anticoagulant according to any one of claims 8 to 10, wherein the first partial reagent and the third partial reagent contain an activator. The reagent kit for detecting lupus anticoagulant according to claim 11, wherein the activator is selected from the group consisting of ellagic acid, kaolin, celite, and colloidal silica. The reagent kit for detecting lupus anticoagulant according to any one of claims 8 to 10, wherein the first partial reagent and the third partial reagent contain snake venom. The reagent kit for detecting lupus anticoagulant according to any one of claims 8 to 10, wherein the first partial reagent and the third partial reagent contain a tissue factor. The reagent kit for detecting lupus anticoagulant according to any one of claims 8 to 14, wherein the second partial reagent and the fourth partial reagent contain calcium ions. The reagent kit for detecting lupus anticoagulant according to any one of claims 1 to 15, wherein the phospholipid is a synthetic phospholipid or a naturally-derived phospholipid purified to a purity of 99% or more.

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