JP4526152B2 - Prothrombin time measuring reagent - Google Patents
Prothrombin time measuring reagent Download PDFInfo
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- JP4526152B2 JP4526152B2 JP2000063945A JP2000063945A JP4526152B2 JP 4526152 B2 JP4526152 B2 JP 4526152B2 JP 2000063945 A JP2000063945 A JP 2000063945A JP 2000063945 A JP2000063945 A JP 2000063945A JP 4526152 B2 JP4526152 B2 JP 4526152B2
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- prothrombin time
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Description
【０００１】
【発明の属する技術分野】
本発明は臨床検査に用いられるプロトロンビン時間測定試薬及びプロトロンビン時間測定方法に関する。
【０００２】
【従来の技術】
血液凝固能検査の中でプロトロンビン時間測定（ＰＴ）は、血液凝固因子であるＩＩ、Ｖ、ＶＩＩ、Ｘ因子の活性を総合的に検査するものであり、凝固能検査のスクリーニング検査及び経口抗凝固薬の効果判定検査として広く実施されている。ＰＴの測定は、組織因子又は組織因子を含むヒト又は動物由来の組織トロンボプラスチンと適当な濃度のカルシウムイオンが主成分である測定試薬に、被検血漿を加え、血液凝固時間を測定することにより行われる。試薬成分であるヒト又は動物由来の組織トロンボプラスチンは通常脳あるいは胎盤といった臓器から抽出される。抽出源となる臓器には血液凝固因子を含む血液成分が含まれている。このため、血液凝固因子を混在させないで組織トロンボプラスチンを抽出することは困難であり、血液凝固因子の活性を感度よく反映する試薬を調製することは困難であった。とりわけＶＩＩ因子と組織トロンボプラスチンは複合体を形成するため、ＶＩＩ因子活性を感度よく反映する組織トロンボプラスチンを調製することが極めて困難であった。
【０００３】
感度の良い組織因子又は組織トロンボプラスチンを含有する試薬を調製することはこの分野に携わる研究者の最大の課題であり、組織トロンボプラスチンを抽出、調製する方法が今までにも示されていたが（特許公告平３−３９２６７、公表特許公告平３−５０３５３４、公開特許公告平成１０−３３０４００、米国特許３，９８３、００４）、何れも実用的に満足の行くものではなかった。
【０００４】
【解決しようとする課題】
本発明の課題は、血液凝固因子活性を感度良く測定できるプロトロンビン時間測定試薬を提供することである。
【０００５】
【解決する手段】
本発明者らは鋭意研究を重ねた結果、組織因子又は動物器官より抽出した組織トロンボプラスチンとニッケル化合物を混合させることにより、飛躍的に感度の良い試薬すなわち、ＷＨＯ参照品と同等の感度を示すＩＳＩ値が１．５以下であり、さらに、外因系凝固因子に対する感受性が高い試薬を調製できることを見出し本発明を完成させるに至った。また目標とするＩＳＩ値は１．５以下に限定されるもので無く当業者が適宜目的に応じ設定可能である。その他条件についても同様に当業者が適宜目的に応じ設定可能である。
【０００６】
本発明は、リン脂質、組織因子および水溶性のニッケル化合物を含有させることを特徴とするプロトロンビン時間測定試薬に関するものである。上記ニッケル化合物が塩化ニッケル、硝酸ニッケル又は酢酸ニッケルから選ばれる少なくとも１又は２以上の化合物を含有することを特徴とするプロトロンビン時間測定試薬に関するものである。上記化合物の濃度が０．１〜５．０ｍｍｏｌ／Ｌであることを特徴とするプロトロンビン時間測定試薬に関するものである。上記プロトロンビン時間測定試薬を用いることを特徴とするプロトロンビン時間測定方法に関するものである。
【０００７】
組織因子は、ヒト又は動物由来の組織トロンボプラスチンに含まれており、さらに遺伝子組換えにより作成することもできる。組織因子の遺伝子組換えによる作成は、遺伝子組換えにより得られた組織因子タンパク質にリン脂質を加えることにより組織因子としての活性が発現される。また、組織トロンボプラスチンは一般にヒト胎盤又はウサギ或いは牛脳から抽出される。ウサギ脳を用いる場合には、ウサギ脳をアセトンで脱水したアセトン粉末を、適当な緩衝液で抽出される。牛脳もアセトン粉末から抽出したり、新鮮脳をミンチにして直接抽出することも有る。抽出した組織トロンボプラスチンは適当な濃度に調整されて、カルシウムイオン及び安定化、賦型剤等を添加して凍結乾燥されるのが一般的である。場合によっては、液状で保存されることもある。
【０００８】
ニッケル化合物を添加する工程は特に限定されないが、上記の最終調製物また抽出液に含ませておくこともできる。添加する濃度は０．１〜５．０ｍｍｏｌ／Ｌであり、とりわけ０．５〜３．０ｍｍｏｌ／Ｌが好適である。添加するニッケル化合物の種類は特に限定されないが、塩化ニッケル、硝酸ニッケル等の無機化合物及び酢酸ニッケル等の有機ニッケル化合物等の中から選ばれる。
【０００９】
【実施例】
以下に実施例を挙げて本発明を更に説明するが、本発明は実施例に限定されるものではない。
【００１０】
【実施例１】
ウサギ脳をアセトンで脱水したウサギ脳アセトン粉末３ｇを生理食塩水１００ｍＬに懸濁し、４５℃で３０分間撹拌した後、５，０００回転で１０分間遠心分離してその上清をトロンボプラスチン抽出原液とした。抽出原液４容と０．１ｍｍｏｌ／Ｌ ＮａＣｌ、１２０ｍｍｏｌ／Ｌ 乳酸カルシウムを含む５０ｍｍｏｌ／Ｌ ＨＥＰＥＳ−Ｔｒｉｓ緩衝液（ｐＨ７．３５）１容を混合してＰＴ測定試薬とした。調製試薬に１ｍｍｏｌ／Ｌの種々の金属化合物を添加し、コントロール血漿のＰＴを測定し、国際感度指数（ＩＳＩ）及びＶＩＩ因子欠乏血漿のＰＴ比を求めた。その結果を表１に示した。
【００１１】
【表１】
以上の結果、塩化ニッケルが特異的にＩＳＩ値を小さくしＶＩＩ因子欠乏血漿に対するＰＴ比を大きくする効果があり、ＰＴ試薬の感度の改善に有効であることが判った。
【００１３】
【実施例２】
実施例１で用いた無添加のＰＴ試薬に、塩化ニッケル、酢酸ニッケル、硝酸ニッケルを各１ｍｍｏｌ／Ｌ添加してＩＳＩ値及びＶＩＩ因子欠乏血漿のＰＴ比の改善効果を実施例１と同様にコントロール血漿のＰＴを測定して調べた。その結果を表２に示した。
【００１４】
【表２】
以上の結果いずれのニッケル化合物でもＩＳＩ値及びＶＩＩ因子欠乏血漿のＰＴ比の改善効果が認められ、ニッケルイオンがＰＴ試薬の感度の改善に有効であることが判った。
【００１６】
【実施例３】
塩化ニッケルの濃度を変化させて、実施例１と同様に操作して、ニッケルイオンの濃度効果を調べた。その結果を表３に示した。
【００１７】
【表３】
以上の結果、塩化ニッケル濃度の増加と共にＩＳＩ値の低下及びＶＩＩ因子に対する感度、すなわち高感度化効果が観測されたが、５ｍＭを超える濃度では、正常血漿のＰＴが大幅に延長するため適当な濃度として塩化ニッケル０．１〜５．０ｍｍｏｌ／Ｌの範囲で効果的であり、とりわけ０．５〜３．０ｍｍｏｌ／Ｌが好適であることが判った。
【００１９】
【発明の効果】
組織因子又は組織トロンボプラスチンとニッケルイオンを含有させることにより血液凝固活性を感度よく測定可能となった。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a prothrombin time measurement reagent and a prothrombin time measurement method used for clinical examination.
[0002]
[Prior art]
Prothrombin time measurement (PT) in the blood coagulation test is a comprehensive test of the activities of factors II, V, VII, and X, which are blood coagulation factors. It is widely used as a test for determining the effect of drugs. Measurement of PT is performed by adding test plasma to a measurement reagent mainly composed of tissue factor or tissue thromboplastin derived from human or animal containing tissue factor and an appropriate concentration of calcium ion, and measuring the blood coagulation time. Is called. The human or animal-derived tissue thromboplastin, which is a reagent component, is usually extracted from an organ such as the brain or placenta. The organ as the extraction source contains blood components including blood coagulation factors. For this reason, it is difficult to extract tissue thromboplastin without mixing blood coagulation factors, and it is difficult to prepare a reagent that reflects the activity of blood coagulation factors with high sensitivity. In particular, since factor VII and tissue thromboplastin form a complex, it has been extremely difficult to prepare tissue thromboplastin that reflects factor VII activity with high sensitivity.
[0003]
The preparation of a reagent containing tissue factor or tissue thromboplastin with high sensitivity is the biggest problem for researchers in this field, and methods for extracting and preparing tissue thromboplastin have been shown (patents). Neither published publication 3-39267, published patent publication 3-503534, published patent publication Heisei 10-330400, U.S. Patent 3,983,004) were not satisfactory in practice.
[0004]
[Problems to be solved]
An object of the present invention is to provide a prothrombin time measurement reagent can sensitively measuring blood coagulation factor activity.
[0005]
[Solution]
As a result of intensive studies, the present inventors have mixed tissue thromboplastin extracted from tissue factor or animal organs with a nickel compound, so that the sensitivity is dramatically improved, that is, an ISI that exhibits the same sensitivity as a WHO reference product. The present invention was completed by finding that a reagent having a value of 1.5 or less and having high sensitivity to an exogenous coagulation factor can be prepared. Further, the target ISI value is not limited to 1.5 or less, and can be appropriately set according to the purpose by those skilled in the art. Other conditions can be similarly set according to the purpose by those skilled in the art.
[0006]
The present invention relates to a prothrombin time measurement reagent comprising a phospholipid, tissue factor and a water-soluble nickel compound. The present invention relates to a prothrombin time measuring reagent, wherein the nickel compound contains at least one compound selected from nickel chloride, nickel nitrate or nickel acetate. The present invention relates to a prothrombin time measurement reagent characterized in that the concentration of the compound is 0.1 to 5.0 mmol / L. The present invention relates to a prothrombin time measuring method characterized by using the above-mentioned prothrombin time measuring reagent .
[0007]
Tissue factor is contained in human or animal-derived tissue thromboplastin, and can also be prepared by genetic recombination. In the preparation of tissue factor by gene recombination, the activity as a tissue factor is expressed by adding phospholipid to the tissue factor protein obtained by gene recombination. Tissue thromboplastin is generally extracted from human placenta or rabbit or bovine brain. When rabbit brain is used, acetone powder obtained by dehydrating rabbit brain with acetone is extracted with an appropriate buffer. Cattle brain can also be extracted from acetone powder, or fresh brain can be minced directly. In general, the extracted tissue thromboplastin is adjusted to an appropriate concentration and freeze-dried by adding calcium ions, stabilizing agents, excipients, and the like. In some cases, it may be stored in liquid form.
[0008]
The step of adding the nickel compound is not particularly limited, but can be included in the final preparation or the extract. The concentration to be added is 0.1 to 5.0 mmol / L, and 0.5 to 3.0 mmol / L is particularly preferable. The kind of nickel compound to be added is not particularly limited, but is selected from inorganic compounds such as nickel chloride and nickel nitrate and organic nickel compounds such as nickel acetate.
[0009]
【Example】
EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to the examples.
[0010]
[Example 1]
Rabbit brain dehydrated with acetone 3 g of rabbit brain acetone powder was suspended in 100 mL of physiological saline, stirred at 45 ° C. for 30 minutes, centrifuged at 5,000 rpm for 10 minutes, and the supernatant was used as a thromboplastin extraction stock solution. . 4 volumes of the extraction stock solution and 1 volume of 50 mmol / L HEPES-Tris buffer (pH 7.35) containing 0.1 mmol / L NaCl and 120 mmol / L calcium lactate were mixed to obtain a PT measurement reagent. Various metal compounds of 1 mmol / L were added to the prepared reagent, PT of control plasma was measured, and international sensitivity index (ISI) and PT ratio of factor VII-deficient plasma were determined. The results are shown in Table 1.
[0011]
[Table 1]
As a result, it was found that nickel chloride has the effect of specifically decreasing the ISI value and increasing the PT ratio for factor VII-deficient plasma, and is effective in improving the sensitivity of the PT reagent.
[0013]
[Example 2]
In the same manner as in Example 1, the effect of improving the ISI value and the PT ratio of factor VII-deficient plasma was controlled by adding 1 mmol / L each of nickel chloride, nickel acetate and nickel nitrate to the additive-free PT reagent used in Example 1. Plasma PT was measured and examined. The results are shown in Table 2.
[0014]
[Table 2]
As a result, any of the nickel compounds was found to improve the ISI value and the PT ratio of factor VII-deficient plasma, and nickel ions were found to be effective in improving the sensitivity of the PT reagent.
[0016]
[Example 3]
By changing the concentration of nickel chloride and operating in the same manner as in Example 1, the concentration effect of nickel ions was examined. The results are shown in Table 3.
[0017]
[Table 3]
As a result, a decrease in ISI value and sensitivity to factor VII, that is, a sensitivity enhancement effect was observed with an increase in nickel chloride concentration. As a result, it was found that nickel chloride was effective in the range of 0.1 to 5.0 mmol / L, and 0.5 to 3.0 mmol / L was particularly preferable.
[0019]
【The invention's effect】
By containing tissue factor or tissue thromboplastin and nickel ions, blood coagulation activity can be measured with high sensitivity.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000063945A JP4526152B2 (en) | 2000-03-08 | 2000-03-08 | Prothrombin time measuring reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000063945A JP4526152B2 (en) | 2000-03-08 | 2000-03-08 | Prothrombin time measuring reagent |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2001255332A JP2001255332A (en) | 2001-09-21 |
| JP2001255332A5 JP2001255332A5 (en) | 2007-04-05 |
| JP4526152B2 true JP4526152B2 (en) | 2010-08-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2000063945A Expired - Fee Related JP4526152B2 (en) | 2000-03-08 | 2000-03-08 | Prothrombin time measuring reagent |
Country Status (1)
| Country | Link |
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| JP (1) | JP4526152B2 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4377207B2 (en) * | 2003-11-28 | 2009-12-02 | シスメックス株式会社 | Blood coagulation time measurement method and blood coagulation time measurement reagent |
| JP2008088103A (en) * | 2006-09-30 | 2008-04-17 | Sysmex Corp | Reagent for measuring factor ii, vii, ix and x and method for producing the same |
| JP4829828B2 (en) | 2007-03-28 | 2011-12-07 | シスメックス株式会社 | Reagent for measuring blood coagulation and method for stabilizing tissue factor |
| JP6595247B2 (en) * | 2015-07-30 | 2019-10-23 | シスメックス株式会社 | Coagulation time measurement method and apparatus, coagulation time measurement reagent and reagent kit |
| JP6626761B2 (en) | 2016-03-30 | 2019-12-25 | シスメックス株式会社 | Prothrombin time measuring reagent and method for producing the same |
| EP3859327A4 (en) | 2018-09-25 | 2022-09-07 | Sekisui Medical Co., Ltd. | Method for measuring blood coagulation time |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5991899A (en) * | 1982-09-29 | 1984-05-26 | オ−ソ・ダイアグノステイツク・システムズ・インコ−ポレ−テツド | Thromboplastin reagent |
| JPS62239989A (en) * | 1986-04-11 | 1987-10-20 | Sekisui Chem Co Ltd | Promoter for hydrolase activity |
| JPS62240616A (en) * | 1986-04-11 | 1987-10-21 | Sekisui Chem Co Ltd | Promoter for blood coagulation |
| JPS6383669A (en) * | 1986-09-29 | 1988-04-14 | Sekisui Chem Co Ltd | Blood agglutination accelerator |
| JPS6383670A (en) * | 1986-09-29 | 1988-04-14 | Sekisui Chem Co Ltd | Vessel for blood inspection |
| JPH0339267B2 (en) * | 1981-12-21 | 1991-06-13 | Boehringer Mannheim Gmbh | |
| JPH05506309A (en) * | 1990-04-17 | 1993-09-16 | アナリティカル・コントロール・システムズ・インコーポレーテッド | Coagulation assays and reagents |
| JPH11514101A (en) * | 1997-04-23 | 1999-11-30 | インストルメンテーション ラボラトリー エス．ピー．アー． | Prothrombin time reagent based on recombinant rabbit tissue factor |
-
2000
- 2000-03-08 JP JP2000063945A patent/JP4526152B2/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0339267B2 (en) * | 1981-12-21 | 1991-06-13 | Boehringer Mannheim Gmbh | |
| JPS5991899A (en) * | 1982-09-29 | 1984-05-26 | オ−ソ・ダイアグノステイツク・システムズ・インコ−ポレ−テツド | Thromboplastin reagent |
| JPS62239989A (en) * | 1986-04-11 | 1987-10-20 | Sekisui Chem Co Ltd | Promoter for hydrolase activity |
| JPS62240616A (en) * | 1986-04-11 | 1987-10-21 | Sekisui Chem Co Ltd | Promoter for blood coagulation |
| JPS6383669A (en) * | 1986-09-29 | 1988-04-14 | Sekisui Chem Co Ltd | Blood agglutination accelerator |
| JPS6383670A (en) * | 1986-09-29 | 1988-04-14 | Sekisui Chem Co Ltd | Vessel for blood inspection |
| JPH05506309A (en) * | 1990-04-17 | 1993-09-16 | アナリティカル・コントロール・システムズ・インコーポレーテッド | Coagulation assays and reagents |
| JPH11514101A (en) * | 1997-04-23 | 1999-11-30 | インストルメンテーション ラボラトリー エス．ピー．アー． | Prothrombin time reagent based on recombinant rabbit tissue factor |
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| Publication number | Publication date |
|---|---|
| JP2001255332A (en) | 2001-09-21 |
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