JP4467407B2 - Lupus Anticoagulant Detection Reagent Kit - Google Patents

Description

  The present invention relates to a reagent kit capable of specifically detecting lupus anticoagulant positive disease by in vitro blood test.

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, etc., and the time until coagulation is prolonged due to a phospholipid-dependent coagulation reaction. It is believed to be an autoantibody against phospholipids.
It has recently 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 and inhibits the conversion of prothrombin to thrombin.

The LA detection reagent having such characteristics includes a first coagulation time measurement reagent containing a phosphatidylserine-containing phospholipid and a second coagulation time measurement reagent containing a phosphatidylserine-containing phospholipid. The ratio of the phosphatidylserine contained in the time measurement reagent to the total phospholipid is different from the ratio of the phosphatidylserine contained in the second coagulation time measurement reagent to the total phospholipid, and the phosphatidylserine concentration of the first coagulation time measurement reagent is A lupus anticoagulant measuring reagent characterized by being higher than the two coagulation time measuring reagent is known (Patent Document 1). Patent Document 1 discloses an LA measurement reagent that can be distinguished from heparin-administered patient plasma and warfarin-administered patient plasma.
On the other hand, a reagent having high detection sensitivity capable of detecting even LA weak positive specimens is desired in the LA test, but Patent Document 1 does not describe improvement of detection sensitivity for LA weak positive specimens.

JP 2004-69697 A

  An object of the present invention is to provide a reagent kit that can enhance the detection sensitivity of LA and can specifically detect even a weak LA positive sample.

  The present invention is a lupus anticoagulant detection reagent kit comprising a first coagulation time measurement reagent and a second coagulation time measurement reagent, wherein the first coagulation time measurement reagent comprises phosphatidylserine as a phospholipid, calcium ion, It contains at least one non-human-derived substance selected from the group consisting of vertebrate antibodies other than humans, serum, plasma and immunoglobulins, and the second clotting time measurement reagent is calcium ion and first clotting time measurement. The present invention relates to a lupus anticoagulant detection reagent kit characterized by containing a lower concentration of phosphatidylserine than the reagent.

  In the reagent kit of the present invention, a difference in coagulation time can be recognized specifically with respect to LA, and only LA positive patients can be detected with high sensitivity.

  The detection reagent kit of the embodiment of the present invention is a reagent kit that can specifically detect lupus anticoagulant in a blood or plasma sample even if it is a weak positive sample.

  The detection reagent kit of an embodiment of the present invention is a lupus anticoagulant detection reagent kit comprising a first clotting time measuring reagent and a second clotting time measuring reagent, wherein the first clotting time measuring reagent is phosphatidyl as a phospholipid. Containing serine, calcium ions, and at least one non-human substance selected from the group consisting of vertebrate antibodies other than humans, serum, plasma and immunoglobulins, and the second clotting time measurement reagent comprises calcium ions And a combination of two kinds of coagulation time measurement reagents containing a lower concentration of phosphatidylserine than the first coagulation time measurement reagent.

  The phosphatidylserine (hereinafter sometimes abbreviated as PS) contained as a phospholipid in the first coagulation time measurement reagent and the second coagulation time measurement reagent may be a synthetic phosphatidylserine or a naturally-derived phosphatidylserine. It may be. The phosphatidylserine is preferably synthetic phosphatidylserine. When natural phosphatidylserine is used, it is preferable to use one purified to a purity of 99% or more.

  Examples of phospholipids other than phosphatidylserine include phosphatidylethanolamine (hereinafter abbreviated as PE) and phosphatidylcholine (hereinafter abbreviated as PC).

  As a non-human vertebrate antibody, serum, plasma, or immunoglobulin contained to capture lupus anticoagulant (hereinafter referred to as “non-human-derived substance” when they are not distinguished and collectively referred to) Are preferably mammalian antibodies other than humans and pigs, serum or immunoglobulins, specifically mouse IgG, horse IgG, bovine IgG, mouse serum, horse serum, bovine serum, mouse, horse or bovine. α-globulin and the like can be used. Commercially available products include HBR (Heterophile Blocking Reagent) manufactured by Scantibodies, MAK33 (Roche), and NM8. HBR is a reagent added to a test system for the purpose of suppressing interference caused by heterophilic antibodies in an immunoassay method. The HBR binds specifically to LA by the present inventor and is also contained in a blood sample. It was found that it does not bind to components related to blood coagulation time other than LA, and it can be used as an LA antibody capturing agent in the coagulation time measurement reagent of the present invention.

  As a non-human-derived substance contained for capturing LA, only one animal-derived antibody or the like may be used, or antibodies derived from two or more different animals, such as mouse IgG and horse IgG. You may mix and use.

  The second clotting time measurement reagent does not substantially contain a non-human-derived substance serving as an LA capture agent, and preferably does not contain a non-human-derived substance. Therefore, in an LA positive sample, due to the presence of LA, The clotting time is extended. On the other hand, when the first coagulation time measurement reagent and the sample are brought into contact with each other, LA is captured by the non-human-derived substance contained in the first coagulation time measurement reagent. Prolongation of the coagulation time due to the presence of is prevented. Therefore, there is a significant difference between the coagulation time when using the first coagulation time measurement reagent (first coagulation time) and the coagulation time when using the second coagulation time measurement reagent (second coagulation time). If positive, it is positive for LA, and if there is no significant difference, it can be determined as LA negative.

The first clotting time measuring reagent and the second clotting time measuring 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, human placenta, or recombinants thereof 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 the snake venom measurement time. 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 coagulation time measurement reagent and the second coagulation time measurement reagent may further contain a buffer solution such as Hepes and Tris buffer 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 coagulation composition contained in the first coagulation time measurement reagent and the coagulation composition contained in the second coagulation time measurement reagent must be the same type. That is, when the coagulation system composition contained in the first coagulation time measurement reagent measures the coagulation time based on the measurement principle of activated partial thromboplastin time measurement, the coagulation system composition contained in the second coagulation time measurement reagent. Also measure the clotting time based on the measurement principle of activated partial thromboplastin time measurement.

Further, the detection reagent kit may be composed of 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. Good.
At this time, when measuring the coagulation time based on the measurement principle of the activated partial thromboplastin time measurement, the first partial reagent and the third partial reagent contain a phospholipid and an activator. The partial reagent includes calcium ions.
When measuring the coagulation time based on the principle of snake venom measurement time, the first partial reagent and the third partial reagent contain phospholipid and snake venom, and the second partial reagent and the fourth partial reagent contain calcium ions. Is included.
Furthermore, when measuring the clotting time based on the principle of prothrombin time measurement, the first partial reagent and the third partial reagent contain phospholipid and tissue factor, and the second partial reagent and the fourth partial reagent include calcium. Ions are included.
Note that the first partial reagent may contain a non-human-derived substance.

Further, the detection reagent kit includes a first clotting time measurement reagent comprising a first partial reagent, a second partial reagent and a third partial reagent, and a second clotting time comprising a fourth partial reagent, a fifth partial reagent and a sixth partial reagent. You may make it comprise with a measurement reagent.
At this time, when measuring the coagulation time based on the measurement principle of the activated partial thromboplastin time measurement, the first partial reagent and the fourth partial reagent contain phospholipids, and the second partial reagent and the fifth partial reagent Contains an activator, and the third and sixth partial reagents contain calcium ions.
When measuring the coagulation time based on the principle of snake venom measurement time, the first partial reagent and the fourth partial reagent contain phospholipid, and the second partial reagent and the fifth partial reagent contain snake venom. The third partial reagent and the sixth partial reagent contain calcium ions.
Furthermore, when measuring the coagulation time based on the principle of prothrombin time measurement, the first partial reagent and the fourth partial reagent contain phospholipid, and the second partial reagent and the fifth partial reagent contain tissue factor. The third partial reagent and the sixth partial reagent contain calcium ions.
Note that the first partial reagent may contain a non-human-derived substance.

The content of the phospholipid in the first clotting time measuring reagent and the second clotting time measuring reagent is obtained by mixing an equal amount of the sample with the first clotting time measuring reagent or the second clotting time measuring reagent (50 μL of the sample and the first clotting time). In the measurement method for measuring the coagulation time, the phosphatidylserine content in the first coagulation time measurement reagent is 35 to 370 μM, preferably 60 to 250 μM. The phosphatidylethanolamine content is 40-410 μM, preferably 65-270 μM, and the phosphatidylcholine content is 35-385 μM, preferably 60-255 μM. Further, the content of the non-human-derived substance is preferably 0.1 to 50 mg, preferably 0.2 to 10 mg, more preferably 0.5 to 6 mg per mL of the subject. This is because at a concentration of 0.1 mg or less per 1 mL of the measurement sample, LA is insufficiently captured in the case of an LA positive specimen, and the coagulation time is extended due to the uncaptured LA.
The phosphatidylserine content in the second clotting time measurement reagent is 1 to 25 μM, preferably 5 to 20 μM, the phosphatidylethanolamine content is 1 to 30 μM, preferably 5 to 20 μM, and the phosphatidylcholine content is 10 -150 μM, preferably 25-65 μM.

  The phosphatidylserine can be used within the above-mentioned concentration range, and the ratio of the phosphatidylserine contained in the first clotting time measurement reagent to the total phospholipids is the total phosphatidylserine contained in the second clotting time measurement reagent. What differs from the ratio with respect to a lipid is preferable, and it is preferable that the ratio with respect to the total phospholipid of the phosphatidylserine of a 1st coagulation time measurement reagent is larger than the ratio with respect to the total phospholipid of the phosphatidylserine of a 2nd coagulation time measurement reagent.

  The concentration of the above-mentioned phospholipid is the concentration when 50 μL of the specimen 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 specimen and the clotting time measuring reagent is different, What is necessary is just to make it the final concentration of the phospholipid after mixing a 1st coagulation time measuring reagent or a 2nd coagulation time measuring reagent become the range similar to said case.

When the first coagulation time measurement reagent is a combination of a first partial reagent and a second partial reagent, and the second coagulation time measurement reagent is also a combination of a third partial reagent and a fourth partial reagent, The phospholipid content of the 1st partial reagent and the 3rd partial reagent is equal to the phosphatidyl in the 1st partial reagent in the measurement method in which the sample and the 1st partial reagent or the 3rd partial reagent are mixed in equal amounts and the clotting time is measured. Serine content is phosphatidylserine content 35-370 μM, preferably 60-250 μM, phosphatidylethanolamine content 40-410 μM, preferably 65-270 μM, phosphatidylcholine content 35-385 μM, preferably 60 ~ 255 μM. Further, the content of the non-human-derived substance is preferably 0.1 to 50 mg, preferably 0.2 to 10 mg, more preferably 0.5 to 6 mg per mL of the subject. This is because when the amount is 0.1 mg or less per 1 mL of the measurement sample, LA is not sufficiently captured in the case of an LA sample, and the coagulation time is extended due to the uncaptured LA.
The phosphatidylserine content of the third partial reagent is 1 to 25 μM, preferably 5 to 20 μM, the phosphatidylethanolamine content is 1 to 30 μM, preferably 5 to 20 μM, and the phosphatidylcholine content is 10 to 150 μM. Preferably it is 25-65 micromol.

The first clotting time measuring reagent is a combination of a first partial reagent, a second partial reagent and a third partial reagent, and the second clotting time measuring reagent is also a fourth partial reagent, a fifth partial reagent and a sixth partial reagent. In the measurement method in which the phospholipid content of the first partial reagent and the fourth partial reagent is mixed with an equal amount of the sample and the first partial reagent or the fourth partial reagent, and the coagulation time is measured. The phosphatidylserine content in the first partial reagent is 35 to 370 μM, preferably 60 to 250 μM, the phosphatidylethanolamine content is 40 to 410 μM, preferably 65 to 270 μM, and the phosphatidylcholine content is 35 to 385 μM. Preferably it is 60-255 micromol. Further, the content of the non-human-derived substance is preferably 0.1 to 50 mg, preferably 0.2 to 10 mg, more preferably 0.5 to 6 mg per mL of the subject. This is because at a concentration of 0.1 mg or less per 1 mL of the measurement sample, LA is insufficiently captured in the case of an LA positive specimen, and the coagulation time is extended due to the uncaptured LA.
The phosphatidylserine content of the fourth partial reagent is 1 to 25 μM, preferably 5 to 20 μM, the phosphatidylethanolamine content is 1 to 30 μM, preferably 5 to 20 μM, and the phosphatidylcholine content is 10 to 150 μM. Preferably it is 25-65 micromol.

Next, a method for detecting LA using the reagent kit of the present invention having the above configuration will be described.
First, divide the specimen sample into two parts, add the first clotting time measurement reagent containing high concentration PS to one, and add the second clotting time measurement reagent containing low concentration PS to the other specimen sample. To do.
When the first clotting time measurement reagent is divided into the first partial reagent and the second partial reagent, the sample and the first partial reagent are mixed and then the second partial reagent is added.
Further, when the first coagulation time measurement reagent is divided into the first partial reagent, the second partial reagent, and the third partial reagent, the sample and the first partial reagent are mixed, and the second partial reagent, the third partial reagent are mixed. Add reagents in the order of reagents.
After each reagent is added to the specimen, the clotting time of the sample is measured. The measurement of the coagulation time may be performed by a method known to those skilled in the art, or may be performed using a known automatic measuring device.

  Here, as a sample used for LA detection, it is preferable to use plasma rather than using blood as it is, and it is more preferable to use platelet-free plasma. Moreover, you may use what mixed normal plasma or normal platelet removal normal plasma into the sample. This is because mixing normal plasma makes it possible to prevent the prolongation of clotting time based on clotting factor deficiency and increases the sensitivity of LA. When mixing the sample and normal plasma, the mixing ratio 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 a sample derived from an LA positive patient, coagulation when using a second coagulation time measurement reagent containing a low concentration of PS than using a first coagulation time measurement reagent containing a high concentration of PS. The time is longer. On the other hand, in samples derived from blood coagulation factor-deficient patients, warfarin-administered patients, and heparin-administered patients, no significant difference in coagulation time is 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 between the first clotting time measuring reagent and the second clotting time measuring reagent, but in order to more accurately determine the clotting time of the patient-derived sample relative to the clotting time of normal plasma. Ratio, for example, the Rossner Index (E. Rosner, et al., Thromb. Haemast. 1987, 57: 144-149) or Lupus Ratio (LR) (R. Schjetlein, et al., Thromb. Res. 1993, 69: 239-250), which is based on the same principle as LR, but it is preferable to make a determination based on a clotting time ratio in which the specimen and normal plasma are not mixed.
In the case of normal plasma, since the PS content does not affect the clotting time, the LR value corresponding to the ratio of the first clotting time reagent and the second clotting time reagent is about 1. In addition, even when derived from blood coagulation factor-deficient patients, warfarin-treated patients, or heparin-treated patients, the clotting time is prolonged compared to normal plasma, but there is almost no difference in clotting time due to the difference in PS concentration. Therefore, the LR value is about 1. On the other hand, in the case of LA positive, the second clotting time measurement reagent measures the first clotting time measurement depending on the concentration and combination of the first clotting time measuring reagent and the second clotting time measuring reagent. Since the clotting time is longer than that of the reagent, the sample from the LA positive patient can be automatically detected by comparing the LR values.

The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
(Comparative Example 1)

A lupus anticoagulant measuring reagent (2 reagent system) based on the measurement principle of activated partial thromboplastin time measurement was prepared by the method shown below.
That is, LA-H consists of a first partial reagent and a second partial reagent, LA-L consists of a third partial reagent and a fourth partial reagent, and the concentration of each component of the first partial reagent is 50 mM Hepes. Ellagic acid is 0.1 mM, Tris is 25 mM, PS is 123 μM, PE is 134 μM, PC is 127 μM, and the pH after addition of each component is 7.35. The concentration of each component in the third partial reagent is 50 mM for Hepes, 0.1 mM for ellagic acid, 25 mM for Tris, 12.3 μM for PS, 13.4 μM for PE, and 38.2 μM for PC. 7.35.
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.

  Prepare 2 sets of normal plasma and 50μL of each sample, add 50μL of 1st partial reagent (or 3rd partial reagent) to each sample in one set, warm to 37 ℃ for 5 minutes, then each sample 50 μL of the second partial reagent (or the fourth partial reagent) was added to and the coagulation time was measured. The coagulation time was measured using a fully automatic blood coagulation analyzer Coagrex 800 (Shimadzu Corporation). The measurement was performed twice to obtain an average value.

  Coagtrol N (manufactured by Sysmex Corporation) is used as normal plasma, and LA positive patient plasma (sample numbers 1 to 21) shown in Table 1 is used as a measurement sample, and healthy human plasma (sample numbers 22 to 29) shown in Table 2 is used. ), Warfarin-administered patient plasma (sample numbers 30-39), factor VIII-deficient patient plasma (sample numbers 40-49) and heparin-administered patient plasma (sample numbers 50-53).

  Healthy human plasma was purchased from George King Biomedical (USA) and Alpha Therapeutic (USA). LA positive patient plasma, warfarin-administered patient plasma, heparin-administered patient plasma, and coagulation factor-deficient patient plasma were purchased from George King Biomedical (USA), Institute of Medical Biology, American Diagnostic Inc (USA), Sanfco.

From the coagulation time measured for each sample, the coagulation time ratio represented by the following formula 1 was calculated.
[Formula 1]
Solidification time ratio = (b / a) / (d / c) = bc / ad
Here, a, b, c, and d in Formula 1 represent measured values obtained by the combinations shown below. That is, a: seconds of measurement sample of LA-H, b: seconds of measurement sample of LA-L, c: seconds of normal plasma of LA-H, d: seconds of normal plasma of LA-L The solidification time ratio of the measurement sample is shown as HBR (-) in Tables 1 and 2. In addition, a coagulation time ratio of 1.6 or more was determined as LA positive (LA +).
(Comparative Example 2)

Coagulation of LA positive patient plasma (Sample Nos. 1 to 21 shown in Table 1) using diluted Russell snake venom reagent (manufactured by Medical and Biological Laboratories) and STACLOT-LA reagent (Roche), which are commercially available LA measurement reagents Time was measured.
Measurement of each LA positive patient plasma using the diluted Russell snake venom reagent was performed by measuring the coagulation time according to the usage volume and calculating the “ratio of coagulation time”. The results are shown in Table 1 as dRVVT.
In addition, each LA positive patient plasma using STACLOT-LA reagent was measured by measuring the clotting time according to the dosage and calculating the “difference in clotting time”.

  The results are shown in Table 1 as STACLOT. When the ratio of coagulation time calculated using diluted Russell snake venom reagent was 1.3 or more, it was judged as LA positive, and when the difference in coagulation time calculated using STACLOT-LA reagent was 8 seconds or more, it was judged as positive.

Example 1
Measurement and determination were performed in the same manner as in Comparative Example 1 except that HBR (manufactured by Scantibodies) was added to the first partial reagent of Comparative Example 1 so as to be 1 mg / mL. The results are shown as HBR (+) in Tables 1 and 2.

表1に示すようにdRVVT試薬およびSTACLOT試薬とも陽性である検体（検体番号1〜13）はHBR(-)、HBR(+)とも陽性であったが、dRVVTおよびSTACLOTのいずれか一方が陽性である弱陽性検体（検体番号14〜21）につてはHBR(-)の試薬では2検体のみ陽性であったが、HBR(+)の試薬は全て陽性であり、LAが弱陽性であっても検出することが明らかとなった。また、表２に示す健常人血漿（検体番号22〜29）、ワーファリン投与患者血漿（検体番号30〜39）、第ＶＩＩＩ因子欠乏血漿（検体番号40〜49）およびヘパリン投与患者血漿（検体番号50〜53）につてはHBR(-)、HBR(+)とも陰性と判定され特異的にLAを検出することが判明した。

As shown in Table 1, specimens that were positive for both dRVVT reagent and STACLOT reagent (Sample Nos. 1 to 13) were positive for both HBR (-) and HBR (+), but either dRVVT or STACLOT was positive. For some weak positive specimens (specimen numbers 14-21), only 2 specimens were positive with the HBR (-) reagent, but all HBR (+) reagents were positive and LA was weakly positive. It became clear to detect. In addition, healthy human plasma (sample numbers 22 to 29), warfarin-administered patient plasma (sample numbers 30 to 39), factor VIII-deficient plasma (sample numbers 40 to 49), and heparin-administered patient plasma (sample number 50) shown in Table 2 -53) were determined to be negative for both HBR (-) and HBR (+) and were found to specifically detect LA.

(Example 2)
A coagulation time measurement reagent (3-reagent system) based on the measurement principle of activated partial thromboplastin time measurement was prepared by the method shown below, and a two-reagent system based on the measurement principle of activated partial thromboplastin time measurement prepared in Example 1 was used. The correlation with the reagent was examined.

That is, LA-H consists of a first partial reagent, a second partial reagent and a third partial reagent, and LA-L consists of a fourth partial reagent, a fifth partial reagent and a sixth partial reagent. The concentration of each component of the first partial reagent is 50 mM for Hepes, 25 mM for Tris, 123 μM for PS, 134 μM for PE, 127 μM for PC, 1 mg / mL for HBR (Scantibodies), and the pH after addition of each component is 7.35. The concentration of each component of the fourth partial reagent is 50 mM for Hepes, 25 mM for Tris, 12.3 μM for PS, 13.4 μM for PE, and 38.2 μM for PC, and the pH after addition of each component is 7.35.
Moreover, what was prepared with purified water so that an ellagic acid density | concentration might be set to 0.1 mM was used as the 2nd partial reagent and the 5th partial reagent.
Moreover, what was prepared with purified water so that a calcium chloride concentration might be set to 25 mM was made into the 3rd partial reagent and the 6th partial reagent.

Prepare two sets of measurement samples of 50 μL each, add 50 μL of the first partial reagent (or the fourth partial reagent) to each sample in one set, heat at 37 ° C. for 1 minute, and then add the second partial reagent (or 50 μL of the 5th partial reagent) was added and heated at 37 ° C. for 5 minutes. Further, 50 μL of the 3rd partial reagent (or the 6th partial reagent) was added, and the fully automated blood coagulation analyzer Core Grex 800 (Shimadzu) Coagulation time was measured using Seisakusho Co., Ltd., and the ratio of coagulation time was calculated.
A total of 50 samples including 18 samples of LA positive patient plasma and 32 samples of healthy human plasma were used as the measurement samples used for examining the correlation.

FIG. 1 shows the correlation between the two-reagent reagent and the three-reagent reagent obtained by measuring the same specimen as described above using the two-reagent reagent prepared in Example 1.
As shown in FIG. 1, there was a correlation between the two-reagent reagent and the three-reagent reagent, and it became clear that the three-reagent reagent can be used in the same manner as the two-reagent reagent.

  As described above, it is possible to provide a reagent kit that can be specifically detected even if LA is a weakly positive sample, and can contribute to the field of clinical tests.

It is a figure which shows the correlation of the coagulation time measurement reagent of 3 reagent type | system | groups based on the measurement principle of the activated partial thromboplastin time measurement of Example 2, and the coagulation time measurement reagent of 2 reagent type | system | groups.

Claims (18)

In the lupus anticoagulant detection reagent kit comprising the first clotting time measuring reagent and the second clotting time measuring reagent,
The first clotting time measurement reagent contains phosphatidylserine as a phospholipid, calcium ion, and HBR (Heterophile Blocking Reagent) ,
The lupus anticoagulant detection reagent kit, wherein the second coagulation time measurement reagent contains calcium ions and a lower concentration of phosphatidylserine than the first coagulation time measurement reagent.   The lupus anticoagulant detection reagent kit according to claim 1, wherein the first and second clotting time measuring reagents contain phosphatidylethanolamine and phosphatidylcholine as phospholipids.   The ratio of the phosphatidylserine contained in the first clotting time measurement reagent to the total phospholipid is different from the ratio of the phosphatidylserine contained in the second clotting time measurement reagent to the total phospholipid. Lupus anticoagulant detection reagent kit.   The lupus anticoagulant detection reagent kit 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.   The lupus anticoagulant detection reagent kit 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.   The lupus anticoagulant detection reagent kit 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. The first clotting time measuring reagent comprises a first partial reagent containing phosphatidylserine and HBR (Heterophile Blocking Reagent) and a second partial reagent containing calcium ions, and the second clotting time measuring reagent is phosphatidyl. The lupus anticoagulant detection reagent kit according to any one of claims 1 to 3, comprising a third partial reagent containing serine and a fourth partial reagent containing calcium ions. The first coagulation time measurement reagent comprises a first partial reagent containing phosphatidylserine, an activator and HBR (Heterophile Blocking Reagent) , and a second partial reagent containing calcium ions, and the second coagulation time measurement The lupus anticoagulant detection reagent kit according to claim 4, wherein the reagent comprises a third partial reagent containing phosphatidylserine and an activator and a fourth partial reagent containing calcium ions. The first clotting time measuring reagent is composed of a first partial reagent containing phosphatidylserine, snake venom and HBR (Heterophile Blocking Reagent) , and a second partial reagent containing calcium ions, and the second clotting time measuring reagent is The lupus anticoagulant detection reagent kit according to claim 5, comprising a third partial reagent containing phosphatidylserine and snake venom and a fourth partial reagent containing calcium ions. The first coagulation time measurement reagent comprises a first partial reagent containing phosphatidylserine, tissue factor and HBR (Heterophile Blocking Reagent) , and a second partial reagent containing calcium ions, and the second coagulation time measurement reagent The reagent kit for detecting lupus anticoagulant according to claim 6, comprising a third partial reagent containing phosphatidylserine and tissue factor and a fourth partial reagent containing calcium ions.   The lupus anticoagulant detection reagent kit according to any one of claims 7 to 10, wherein the first partial reagent and the third partial reagent contain phosphatidylethanolamine and phosphatidylcholine.   The first clotting time measurement reagent comprises a first partial reagent containing phosphatidylserine and a non-human-derived substance, a second partial reagent containing an activator, and a third partial reagent containing calcium ions, The lupus according to claim 4, wherein the second clotting time measurement reagent comprises a fourth partial reagent containing phosphatidylserine, a fifth partial reagent containing an activator, and a sixth partial reagent containing calcium ions. Anticoagulant detection reagent kit. The first clotting time measurement reagent comprises a first partial reagent containing phosphatidylserine and HBR (Heterophile Blocking Reagent) , a second partial reagent containing snake venom, and a third partial reagent containing calcium ions, The lupus anticore according to claim 5, wherein the second clotting time measurement reagent comprises a fourth partial reagent containing phosphatidylserine, a fifth partial reagent containing snake venom, and a sixth partial reagent containing calcium ions. Grant detection reagent kit. The first clotting time measurement reagent comprises a first partial reagent containing phosphatidylserine and HBR (Heterophile Blocking Reagent) , a second partial reagent containing tissue factor, and a third partial reagent containing calcium ions. The lupus according to claim 6, wherein the second clotting time measurement reagent comprises a fourth partial reagent containing phosphatidylserine, a fifth partial reagent containing tissue factor, and a sixth partial reagent containing calcium ions. Anticoagulant detection reagent kit.   The lupus anticoagulant detection reagent kit according to any one of claims 12 to 14, wherein the first partial reagent and the fourth partial reagent contain phosphatidylethanolamine and phosphatidylcholine.   The lupus anticoagulant detection reagent kit according to claim 4, 8 or 12, wherein the activator is one or more activators selected from the group consisting of ellagic acid, kaolin, celite and colloidal silica.   The lupus anticoagulant detection reagent kit according to claim 5, 9 or 13, wherein the snake venom is one or more snake venoms selected from the group consisting of Russell snake venom, textarin snake venom and ecarin snake venom.   The lupus anticoagulant detection reagent kit according to claim 6, 10 or 14, wherein the tissue factor is derived from rabbit brain or human placenta.

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