WO1999038997A1 - Method for measuring heparin cofactor ii activity and reagent kit - Google Patents

Method for measuring heparin cofactor ii activity and reagent kit Download PDF

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Publication number
WO1999038997A1
WO1999038997A1 PCT/JP1999/000291 JP9900291W WO9938997A1 WO 1999038997 A1 WO1999038997 A1 WO 1999038997A1 JP 9900291 W JP9900291 W JP 9900291W WO 9938997 A1 WO9938997 A1 WO 9938997A1
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activity
thrombin
measuring
heparin cofactor
hcii
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PCT/JP1999/000291
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French (fr)
Japanese (ja)
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Takashi Goto
Naomi Asahara
Akimasa Omizu
Yahiro Uemura
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Yoshitomi Pharmaceutical Industries, Ltd.
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Priority to AU19836/99A priority Critical patent/AU1983699A/en
Publication of WO1999038997A1 publication Critical patent/WO1999038997A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen

Definitions

  • the present invention relates to a method for measuring the activity of heparin cofactor II (hereinafter, referred to as “HCII”) and a reagent kit for measuring HCII activity.
  • HCII heparin cofactor II
  • HCII is a single-chain plasma glycoprotein with a molecular weight of about 72 kDa determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and the nucleotide sequence of its cDNA has already been identified (J Biol. Chem., 257, 2162-2169, 1982;
  • Antithrombin III (hereinafter referred to as "AT-III") is mainly known as another protein having an antithrombin action in blood, but HCII has a different action site from AT-III. It is thought to function as a thrombin inhibitor in the media of the blood vessel below the vascular endothelial cells rather than on the vascular endothelial cells. The possibility of application to prevention and treatment has been suggested (JP-A-9-117640).
  • HCII plays an important role in vivo, and a measurement method that can accurately quantify HCII contained in samples such as plasma is required.
  • HCII activity is determined as a thrombin inhibitory activity in the presence of heparin using an AT-III assay system.
  • AT-III is mixed in the sample, the thrombin inhibitory activity of AT-III is also measured as HCII activity, and thus the measured value is higher than the actual HCII activity.
  • An object of the present invention is to provide a method for accurately measuring the activity of H C II in which the effect of the interfering substance AT-III is suppressed.
  • Another object of the present invention is to provide a reagent kit used in the above-described method for measuring HCII activity. Is to provide.
  • the present inventors have noticed that AT-III has an SS bond, but HCII does not have an SS bond, and selectively cuts AT-III by cleaving the SS bond with a reducing agent.
  • the present inventors have found a method for measuring the thrombin inhibitory activity of HCII alone, and have completed the present invention.
  • the present invention relates to the following.
  • a reagent kit for measuring HC II activity comprising a reducing agent, a sulfated polysaccharide, thrombin, a thrombin substrate, a reaction terminator, and an HC II standard.
  • FIG. 1 is a diagram showing a calibration curve of Example 2.
  • examples of the sample to be measured include blood, plasma, serum, fraction components obtained therefrom, and other biologically derived liquid components. Those obtained by diluting these specimens with a buffer or the like can also be used.
  • the reducing agent used in the present invention is not particularly limited as long as it can cleave the SS bond. For example, dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, cysteine and the like can be mentioned.
  • the concentration of the reducing agent in the reaction solution in the treatment step with the reducing agent is usually from 0.001 to 10 OmM, preferably from 0.1 to 1 OmM.
  • the treatment step with a reducing agent is preferably performed in a buffer solution having a pH of 7 to 9.
  • the buffer is not particularly limited, and includes, for example, a Tris-HCl buffer, a phosphate buffer and the like.
  • the concentration of the reducing agent in the reaction solution used for measuring the activity is preferably 0.1 mM or less.
  • the reaction between HC II and thrombin is performed in the presence of sulfated polysaccharide.
  • the thrombin inhibitory activity of HC II is enhanced in the presence of sulfated polysaccharide.
  • the sulfated polysaccharides used in the present invention is vulcanized acid - a polysaccharide having (OS 0 3 H), for example, heparin, dermatan sulfate, chondroitin polysulfate, dextran sulfate and the like to. Heparin or dermatan sulfate is preferably used. In particular, dermatan sulfate is preferred because it can specifically activate HC II and is less affected by AT-III.
  • the concentration of the sulfated polysaccharide in the reaction solution is preferably from 0.01 to 10 mg Zm1, and particularly preferably from 0.1 to 1 mg / m1.
  • the origin of the thrombin used in the present invention is not particularly limited. Thrombin from humans, pests, pomas, goats, etc. can be used.
  • the concentration of thrombin in the reaction solution is preferably from 0.01 to 5 UZm1.
  • the measurement of the HCII activity of the present invention is preferably performed in a reaction solution having a low salt concentration.
  • the reaction between thrombin and HC II quickly reaches a steady state when performed in a low salt concentration reaction solution.
  • a low salt concentration means that sodium chloride is not contained or the sodium chloride concentration is 0.25 M or less. Preferably, it does not contain sodium chloride or has a sodium chloride concentration of 0.2 M or less.
  • the reaction between thrombin and H C II is performed in a reaction solution to which sodium chloride has not been added.
  • the treatment step with a reducing agent and the step of measuring residual thrombin activity may be performed in a reaction solution to which sodium chloride has not been added.
  • thrombin substrate is added to react with thrombin i substrate. Thereafter, the reaction is stopped by adding a reaction terminator, and the remaining activity of thrombin is measured.
  • thrombin substrate used in the present invention commercially available ones such as S-228 (H-D-phenylaranyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride) can be used. it can.
  • reaction terminator used in the present invention those similar to the AT-III measurement system can be used.
  • An example for example, an aqueous solution of citrate can be used.
  • a reducing agent is added to the sample (or a diluent thereof) and pretreated at 4 to 40 ° C, preferably 20 to 37 ° C, for 5 to 60 minutes.
  • the pretreated reaction solution is appropriately diluted.
  • the sulfated polysaccharide and thrombin are added, and the mixture is reacted at 20 to 40 ° C., preferably 37 ° C. for 0.5 to 15 minutes.
  • a thrombin substrate is added, and the reaction is carried out at 20 to 40 ° C., preferably at 37, for 0.5 to 15 minutes.
  • the reaction is stopped by adding a reaction terminator.
  • the remaining activity of thrombin is measured.
  • the method for measuring thrombin activity is not particularly limited, and a known method can be used.
  • the residual activity of thrombin can be determined by measuring the absorbance at 405 nm.
  • the above reaction temperature and reaction time can be appropriately changed.
  • Prepare a calibration curve by plotting the logarithm of the HCII activity of the diluted standard with the logarithm of the inhibitory activity of each diluted standard. From the calibration curve obtained, determine the HCII activity in the sample.
  • the present invention also provides a reagent kit for measuring HC II activity, comprising a reducing agent, a sulfated polysaccharide, thrombin, a thrombin substrate, a reaction terminator, and an HC II standard product.
  • Reagents constituting the reagent kit of the present invention may be dissolved in purified water or a buffer so as to have a predetermined concentration in a reaction solution together with excipients, or diluted to a desired concentration at the time of use, according to a conventional method. It can be in the form of a concentrated solution or a lyophilized product.
  • the reagent kit of the present invention may further contain a buffer for dilution.
  • the buffer solution for dilution preferably has a low salt concentration.
  • the dilution buffer preferably does not contain sodium chloride or has a sodium chloride concentration of 0.25 M or less, and more preferably does not contain sodium chloride or has a sodium chloride concentration of 0 or less. . 2 M or less.
  • a buffer to which sodium chloride is not added is used.
  • Example 1 Inactivation effect of AT-III by treatment with reducing agent
  • AT-III concentration was adjusted to 5 UZm1 with 20 mM Tris-HC1, pH 8 buffer containing 10 mM DTT or no DTT, and DTT treatment was performed at 37 ° C for 30 minutes. .
  • DTT treatment was performed in the presence or absence of 5% human serum albumin (HSA).
  • the DTT-treated sample was diluted with a sample diluent containing no DTT (0.04 M Tris, 0.06 M ethylenediaminetetraacetic acid (EDTA), 0.2% HSA, 0.1 mg / m 1 dermatan sulfate, pH 8.
  • the solution was diluted 100-fold with 56) and used for a thrombin activity measurement system.
  • a human thrombin solution (1. OU / ml) 1001 containing 0.05% ⁇ serum albumin (BSA) and 0.05% polyethylene glycol 6000 (PEG6000). , 37 for 5 minutes.
  • a substrate solution (S-2238, 1.52 mM) 100 ⁇ 1 was added thereto, and the mixture was allowed to stand at 37 for 5 minutes. After the reaction was stopped by adding 1,000 ⁇ l of 2% citric acid, the absorbance at 405 nm (A 405 ) was measured.
  • the antithrombin activity is the inhibition rate of the thrombin activity when only the sample diluent is used, and was determined by the following equation. Thrombin activity of one sample when only sample diluent is used
  • the inhibition rate of antithrombin activity by DTT treatment was determined by the following equation. Table 1 shows the results. When DTT processing is not performed When DTT processing is performed
  • the HCII concentration was adjusted to a constant ratio (1: 2: 4) with 0-chome solution (1001] ⁇ DTT, 20 mM Tris-HC1, pH8), and DTT treatment was performed for 37 and 30 minutes.
  • the DTT-treated reaction solution was added to a sample diluent containing no DTT (0.04 MT ris, 0.06 M EDTA, 0.2% HSA, 0.1 mg / m 1 dermatan sulfate, pH 8.
  • the antithrombin activity of a sample when diluted 100-fold with 56) and subjected to DTT treatment in the same manner as in Example 1 was measured.
  • the HCII standard was diluted with a sample diluent containing no DTT (same as above).
  • the antithrombin activity of each diluted standard was measured in the same manner as in Example 1, and a calibration curve was prepared.
  • Fig. 1 shows the calibration curve.
  • the antithrombin activity of the DTT-treated sample was introduced into this calibration curve, and the HCII activity of the DTT-treated sample was calculated.
  • Table 2 shows the results. 0 11 ⁇ 11 of the sample after processing The activity ratio was the same as the HCII concentration ratio of the specimen without DTT treatment. Therefore, it was shown that -DTT treatment had no effect on HC II activity.
  • Thrombin solution (1. OUZml thrombin, 0.05% BSA, 0.05% PEG6000)
  • a reagent kit for measuring HC II activity comprising the above reagents was obtained.
  • the effect of AT-III can be suppressed and the thrombin inhibitory activity of HCII alone can be accurately measured even for a sample containing AT-III.
  • the measurement method of the present invention is a useful method that allows accurate measurement of HCII activity in a short time by a simple operation.

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Abstract

A method for measuring heparin cofactor II (HCII) activity at a high accuracy. This method comprises treating an HCII-containing specimen with a reducing agent, reacting HCII with thrombin in the presence of a sulfated polysaccharide and measuring the residual thrombin activity to thereby determine the thrombin inhibitory activity of HCII in the specimen. A kit for measuring HCII activity consisting of a reducing agent, a sulfated polysaccharide, thrombin, a thrombin substrate, a reaction ceasing agent and an HCII standard. In the above method, antithrombin-III (AT-III) carrying S-S bond is selectively inactivated by the treatment with the reducing agent. Thus, the thrombin inhibitory activity of HCII alone can be accurately measured even in the case of a specimen contaminated with AT-III.

Description

明細書  Specification
へパリンコファクタ一 IIの活性測定方法および試薬キッ ト  Heparin cofactor-1 II activity measurement method and reagent kit
技術分野  Technical field
本発明はへパリンコファクタ一 II (以下、 「HCII」 という） の活性測定方法 および HC II活性測定用試薬キッ 卜に関する。  The present invention relates to a method for measuring the activity of heparin cofactor II (hereinafter, referred to as “HCII”) and a reagent kit for measuring HCII activity.
背景技術  Background art
HCIIは、 SDSポリアクリルアミ ドゲル電気泳動 （SDS— PAGE) によ り求めた分子量が約 72 kD aの一本鎖の血漿糖蛋白質であり、 その cDNAの 塩基配列も既に同定されている （J. Biol. Chem., 257, 2162-2169， 1982;  HCII is a single-chain plasma glycoprotein with a molecular weight of about 72 kDa determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and the nucleotide sequence of its cDNA has already been identified (J Biol. Chem., 257, 2162-2169, 1982;
Nucleic Acids Res., 14, 1073-1088, 1986 ) 。 H C IIの生理作用は十分解明さ れていないが、 トロンビンを特異的に阻害する抗凝固物質の 1つである。 血中で 抗トロンビン作用を有する他の蛋白質としては、 主にアンチトロンビン III (以 下 「AT— III」 という。 ） が知られているが、 HCIIは AT— III とは作用部 位が異なり、 血管内皮細胞上よりも血管内皮細胞下の血管中膜におけるトロンビ ン阻害物質として機能していると考えられ、 血流異常を生じる各種疾患をはじめ、 敗血症や各種臓器障害、 各種炎症などの疾患の予防および治療への適用可能性が 示唆されている （特開平 9一 1 76040号公報） 。 Nucleic Acids Res., 14, 1073-1088, 1986). The physiological effects of HC II are not fully understood, but it is one of the anticoagulants that specifically inhibits thrombin. Antithrombin III (hereinafter referred to as "AT-III") is mainly known as another protein having an antithrombin action in blood, but HCII has a different action site from AT-III. It is thought to function as a thrombin inhibitor in the media of the blood vessel below the vascular endothelial cells rather than on the vascular endothelial cells. The possibility of application to prevention and treatment has been suggested (JP-A-9-117640).
このように、 HCIIは生体内で重要な役割を果しており、 血漿などの検体中に 含まれる HCIIを正確に定量できる測定方法が必要である。 HCIIの活性は、 A T一 III測定系を用いて、 へパリン存在下でトロンビン阻害活性として求められ る。 しかしながら、 この方法では検体中に AT— IIIが混在している場合、 AT -III のトロンビン阻害活性も HCII活性として測定されるため、 測定値が実際 の HCII活性より高い値となる。  As described above, HCII plays an important role in vivo, and a measurement method that can accurately quantify HCII contained in samples such as plasma is required. HCII activity is determined as a thrombin inhibitory activity in the presence of heparin using an AT-III assay system. However, in this method, when AT-III is mixed in the sample, the thrombin inhibitory activity of AT-III is also measured as HCII activity, and thus the measured value is higher than the actual HCII activity.
発明の開示  Disclosure of the invention
本発明の目的は、 妨害物質である AT— III の影響が抑制された、 精度のよい H C IIの活性測定方法を提供することである。  An object of the present invention is to provide a method for accurately measuring the activity of H C II in which the effect of the interfering substance AT-III is suppressed.
また本発明の目的は、 上記 HCIIの活性測定方法で使用される試薬キッ トを提 供することである。 Another object of the present invention is to provide a reagent kit used in the above-described method for measuring HCII activity. Is to provide.
本発明者らは、 AT— III は S— S結合を有するが、 HCIIには S— S結合が ないことに着目し、 還元剤で S— S結合を切断することにより AT— III を選択 的に不活性化し、 HCIIのみのトロンビン阻害活性を測定する方法を見いだし、 本発明を完成するに至った。  The present inventors have noticed that AT-III has an SS bond, but HCII does not have an SS bond, and selectively cuts AT-III by cleaving the SS bond with a reducing agent. The present inventors have found a method for measuring the thrombin inhibitory activity of HCII alone, and have completed the present invention.
本発明は以下に関する。  The present invention relates to the following.
(1) HCIIを含有する検体を還元剤で処理した後、 硫酸化多糖の存在下で HC IIをトロンビンと反応させ、 残存トロンビン活性を測定することにより検体中の H C IIのトロンビン阻害活性を求めることを特徴とする H C IIの活性測定方法。 (1) After treating a sample containing HCII with a reducing agent, react HCII with thrombin in the presence of sulfated polysaccharide and measure the residual thrombin activity to determine the thrombin inhibitory activity of HCII in the sample A method for measuring the activity of HC II.
(2) 還元剤がジチオトレイ トールである上記 （1) の方法。 (2) The method according to (1) above, wherein the reducing agent is dithiothreitol.
(3) 還元剤による処理工程における反応液中の還元剤の濃度が 0. 00 1 mM 〜10 OmMである上記 （1) または （2) の方法。  (3) The method according to (1) or (2) above, wherein the concentration of the reducing agent in the reaction solution in the treatment step with the reducing agent is 0.001 mM to 10 OmM.
(4) 硫酸化多糖がデルマタン硫酸である上記 （1) 〜 （3) のいずれかに記載 の方法。  (4) The method according to any one of the above (1) to (3), wherein the sulfated polysaccharide is dermatan sulfate.
(5) HCIIとトロンビンの反応を低塩濃度の反応液中で行う上記 （1) 〜 （4) のいずれかに記載の方法。  (5) The method according to any one of (1) to (4), wherein the reaction between HCII and thrombin is performed in a reaction solution having a low salt concentration.
(6) H C IIとトロンビンの反応を塩化ナトリゥムを含有しないかまたは塩化ナ トリウム濃度 0. 25 M以下の反応液中で行う上記 （1) 〜 （4) のいずれかに 記載の方法。  (6) The method according to any one of the above (1) to (4), wherein the reaction between HC II and thrombin is carried out in a reaction solution containing no sodium chloride or having a sodium chloride concentration of 0.25 M or less.
(7) HCIIと卜ロンビンの反応を塩化ナトリゥムを含有しないかまたは塩化ナ トリウム濃度 0. 2 M以下の反応液中で行う上記 （1) 〜 （4) のいずれかに記 載の方法。  (7) The method according to any one of (1) to (4) above, wherein the reaction between HCII and thrombin is carried out in a reaction solution containing no sodium chloride or having a sodium chloride concentration of 0.2 M or less.
(8) 還元剤、 硫酸化多糖、 トロンビン、 トロンビンの基質、 反応停止剤、 およ び H C II標準品からなる H C Π活性測定用試薬キッ ト。  (8) A reagent kit for measuring HC II activity, comprising a reducing agent, a sulfated polysaccharide, thrombin, a thrombin substrate, a reaction terminator, and an HC II standard.
(9) 還元剤がジチオトレイ トールである上記 （8) の HCII活性測定用試薬キ ッ 卜 ο  (9) Reagent kit for measuring HCII activity in (8) above, wherein the reducing agent is dithiothreitol.
(10) 硫酸化多糖がデルマタン硫酸である上記 （8) または （9) の HCII活 性測定用試薬キッ ト。 (10) The HCII activity of (8) or (9) above, wherein the sulfated polysaccharide is dermatan sulfate. Reactivity kit for sex determination.
(1 1) さらに希釈用緩衝液を含む上記 （8) 〜 （10) のいずれかに記載の H C II活性測定用試薬キッ 卜。  (11) The reagent kit for measuring HC II activity according to any one of the above (8) to (10), further comprising a dilution buffer.
(12) 希釈用緩衝液が低塩濃度を有する緩衝液である上記 （1 1) の HCII活 性測定用試薬キッ 卜。  (12) The kit for measuring HCII activity according to (11) above, wherein the dilution buffer has a low salt concentration.
(13) 希釈用緩衝液が塩化ナ卜リゥムを含有しないかまたは塩化ナトリゥム濃 度 0. 25M以下である上記 （1 1) の HCII活性測定用試薬キッ ト。  (13) The reagent kit for measuring HCII activity according to (11) above, wherein the dilution buffer does not contain sodium chloride or has a sodium chloride concentration of 0.25 M or less.
(14) 希釈用緩衝液が塩化ナトリゥムを含有しないかまたは塩化ナ卜リゥム濃 度 0. 2 M以下である上記 （1 1) の HCII活性測定用試薬キッ ト。  (14) The reagent kit for measuring HCII activity according to (11) above, wherein the dilution buffer does not contain sodium chloride or has a sodium chloride concentration of 0.2 M or less.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1は実施例 2の検量線を示す図である。  FIG. 1 is a diagram showing a calibration curve of Example 2.
発明の詳細な説明  Detailed description of the invention
本発明において、 測定対象となる検体としては、 血液、 血漿、 血清およびこれ らから得られた分画成分、 ならびにその他の生体由来の液状成分などが例示され る。 またこれらの検体を緩衝液などで希釈したものを使用することもできる。 本発明で用いる還元剤は、 S - S結合を切断できるものであれば特に限定され ない。 例えば、 ジチオトレイ ト一ル （DTT) 、 ジチォエリ トリ トール（DTE)、 2—メルカプトエタノール、 システィンなどが挙げられる。  In the present invention, examples of the sample to be measured include blood, plasma, serum, fraction components obtained therefrom, and other biologically derived liquid components. Those obtained by diluting these specimens with a buffer or the like can also be used. The reducing agent used in the present invention is not particularly limited as long as it can cleave the SS bond. For example, dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, cysteine and the like can be mentioned.
還元剤による処理工程における反応液中の還元剤の濃度は、 通常 0. 001〜 10 OmMであり、 好ましくは 0. 1〜 1 OmMである。 還元剤による処理工程 は pH 7〜9の緩衝液中で行うことが好ましい。 緩衝液は特に限定されないが、 えば、 トリス—塩酸緩衝液、 リン酸緩衝液などが挙げられる。  The concentration of the reducing agent in the reaction solution in the treatment step with the reducing agent is usually from 0.001 to 10 OmM, preferably from 0.1 to 1 OmM. The treatment step with a reducing agent is preferably performed in a buffer solution having a pH of 7 to 9. The buffer is not particularly limited, and includes, for example, a Tris-HCl buffer, a phosphate buffer and the like.
還元剤で処理した後、 反応液中の還元剤の濃度が 0. ImM以下となるよう希 釈する。 希釈液としては pH6〜l 0の緩衝液、 例えばトリス—塩酸緩衝液、 リ ン酸緩衝液などが挙げられる。 この希釈液中に硫酸化多糖を含有させることもで きる。 還元剤は蛋白変性作用を有するため、 トロンビン活性測定を行う反応液中 に高濃度の還元剤が存在すると活性測定に影響を及ぼす。 したがって、 トロンビ ン活性測定に供する反応液中の還元剤の濃度は 0 . 1 mM以下が好ましい。 After the treatment with the reducing agent, dilute the reaction solution so that the concentration of the reducing agent in the reaction solution is 0. ImM or less. Examples of the diluent include a buffer having a pH of 6 to 10 such as Tris-HCl buffer, phosphate buffer and the like. A sulfated polysaccharide can be contained in this diluent. Since the reducing agent has a protein denaturing action, the presence of a high concentration of the reducing agent in the reaction solution for measuring the thrombin activity affects the activity measurement. Therefore, Thrombi The concentration of the reducing agent in the reaction solution used for measuring the activity is preferably 0.1 mM or less.
H C IIとトロンビンとの反応は硫酸化多糖の存在下で行う。 H C IIのトロンビ ン阻害活性は硫酸化多糖の存在下で促進される。 本発明で用いる硫酸化多糖は硫 酸基 （― O S 0 3 H) を有する多糖であり、 例えば、 へパリン、 デルマタン硫酸、 コンドロイチンポリ硫酸、 デキストラン硫酸などが挙げられる。 へパリンまたは デルマタン硫酸が好適に用いられる。 特に、 H C IIを特異的に活性化することが でき、 A T— III による影響が少ないことから、 デルマタン硫酸が好ましい。 反 応液中の硫酸化多糖の濃度は 0 . 0 1〜 1 0 m g Zm 1が好適であり、 0 . 1〜 1 m g /m 1が特に好適である。 The reaction between HC II and thrombin is performed in the presence of sulfated polysaccharide. The thrombin inhibitory activity of HC II is enhanced in the presence of sulfated polysaccharide. The sulfated polysaccharides used in the present invention is vulcanized acid - a polysaccharide having (OS 0 3 H), for example, heparin, dermatan sulfate, chondroitin polysulfate, dextran sulfate and the like to. Heparin or dermatan sulfate is preferably used. In particular, dermatan sulfate is preferred because it can specifically activate HC II and is less affected by AT-III. The concentration of the sulfated polysaccharide in the reaction solution is preferably from 0.01 to 10 mg Zm1, and particularly preferably from 0.1 to 1 mg / m1.
本発明で用いるトロンビンの由来は特に限定されない。 ヒト、 ゥシ、 ゥマ、 ャ ギ等の由来のトロンビンを使用することができる。 反応液中のトロンビンの濃度 は 0 . 0 1〜5 UZm 1が好適である。  The origin of the thrombin used in the present invention is not particularly limited. Thrombin from humans, pests, pomas, goats, etc. can be used. The concentration of thrombin in the reaction solution is preferably from 0.01 to 5 UZm1.
本発明の H C II活性測定は、 低塩濃度の反応液中で行うことが好ましい。 低塩 濃度の反応液中で行うことにより、 トロンビンと H C IIとの反応が速やかに定常 状態に達する。 低塩濃度とは、 塩化ナトリゥムを含有しないかまたは塩化ナトリ ゥム濃度が 0 . 2 5 M以下であることを意味する。 好ましくは塩化ナトリゥムを 含有しないかまたは塩化ナトリウム濃度が 0 . 2 M以下である。 特に好ましくは 塩化ナトリゥムを添加していない反応液中でトロンビンと H C IIの反応を行う。 さらに、 還元剤による処理工程、 残存トロンビン活性を測定する工程を塩化ナト リウムを添加していない反応液中で行つてもよい。  The measurement of the HCII activity of the present invention is preferably performed in a reaction solution having a low salt concentration. The reaction between thrombin and HC II quickly reaches a steady state when performed in a low salt concentration reaction solution. A low salt concentration means that sodium chloride is not contained or the sodium chloride concentration is 0.25 M or less. Preferably, it does not contain sodium chloride or has a sodium chloride concentration of 0.2 M or less. Particularly preferably, the reaction between thrombin and H C II is performed in a reaction solution to which sodium chloride has not been added. Further, the treatment step with a reducing agent and the step of measuring residual thrombin activity may be performed in a reaction solution to which sodium chloride has not been added.
H C IIをトロンビンと反応させた後、 トロンビンの基質を添加してトロンビン i基質との反応を行う。 その後、 反応停止剤を添加して反応を停止させ、 トロン ビンの残存活性を測定する。  After reacting H C II with thrombin, a thrombin substrate is added to react with thrombin i substrate. Thereafter, the reaction is stopped by adding a reaction terminator, and the remaining activity of thrombin is measured.
本発明で用いるトロンビンの基質としては、 S— 2 2 3 8 (H— D—フエニル ァラニルー L—ピペコリル— L—アルギニル一 p—ニトロァニリ ド ·二塩酸塩） などの市販のものを使用することができる。  As the thrombin substrate used in the present invention, commercially available ones such as S-228 (H-D-phenylaranyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride) can be used. it can.
本発明で用いる反応停止剤は A T— III 測定系と同様のものが使用できる。 例 えば、 クェン酸水溶液を使用することができる。 As the reaction terminator used in the present invention, those similar to the AT-III measurement system can be used. An example For example, an aqueous solution of citrate can be used.
本発明の測定方法の具体的操作について説明する。  The specific operation of the measuring method of the present invention will be described.
検体 （またはその希釈液） に還元剤を添加し、 4〜4 0 °C、 好ましくは 2 0〜 3 7 °Cにて 5〜6 0分間、 前処理する。 前処理した反応液を適宜希釈する。 ここ に硫酸化多糖とトロンビンを添加し、 2 0〜4 0 °C、 好ましくは 3 7 °Cにて 0. 5 〜1 5分間反応させる。 さらにトロンビンの基質を添加し、 2 0〜4 0 °C、 好ま しくは 3 7でにて0 . 5〜1 5分間反応させる。 反応停止剤を添加して反応を停 止させる。 トロンビンの残存活性を測定する。 トロンビンの活性測定方法は特に 限定されず、 公知の手法を用いることができる。 例えば、 基質として S— 2 2 3 8を使用した場合には、 4 0 5 n mにおける吸光度を測定することにより トロン ビンの残存活性を求めることができる。 上記の反応温度、 反応時間は適宜変更し 得る。 H C II標準品またはその希釈液を種々の濃度に希釈し、 上記と同様の操作 を行いトロンビン残存活性を測定する。 希釈標準品の H C II活性の対数に対して、 各希釈標準品の卜口ンビン阻害活性の対数をプロッ トして検量線を作成する。 得 られた検量線より、 検体中の H C II活性を求める。  A reducing agent is added to the sample (or a diluent thereof) and pretreated at 4 to 40 ° C, preferably 20 to 37 ° C, for 5 to 60 minutes. The pretreated reaction solution is appropriately diluted. Here, the sulfated polysaccharide and thrombin are added, and the mixture is reacted at 20 to 40 ° C., preferably 37 ° C. for 0.5 to 15 minutes. Further, a thrombin substrate is added, and the reaction is carried out at 20 to 40 ° C., preferably at 37, for 0.5 to 15 minutes. The reaction is stopped by adding a reaction terminator. The remaining activity of thrombin is measured. The method for measuring thrombin activity is not particularly limited, and a known method can be used. For example, when S-2 238 is used as a substrate, the residual activity of thrombin can be determined by measuring the absorbance at 405 nm. The above reaction temperature and reaction time can be appropriately changed. Dilute the HC1 standard or its diluent to various concentrations, and perform the same operation as above to measure the residual activity of thrombin. Prepare a calibration curve by plotting the logarithm of the HCII activity of the diluted standard with the logarithm of the inhibitory activity of each diluted standard. From the calibration curve obtained, determine the HCII activity in the sample.
また本発明は、 還元剤、 硫酸化多糖、 トロンビン、 トロンビンの基質、 反応停 止剤、 および H C II標準品からなる H C II活性測定用試薬キッ トを提供する。 本 発明の試薬キッ トを構成する各試薬は、 常法に従って、 賦形剤と共に反応液中で 所定の濃度になるように精製水や緩衝液に溶解した形態、 あるいは使用時に所望 の濃度に希釈する濃厚溶液の形態、 あるいは凍結乾燥品の形態とすることができ る。 本発明の試薬キッ トは、 さらに希釈用緩衝液を含んでいてもよい。 該希釈用 辏衝液は低塩濃度であることが好ましい。 詳細には、 希釈用緩衝液は塩化ナ卜リ ゥムを含有しないかまたは塩化ナトリウム濃度 0 . 2 5 M以下であることが好ま しく、 より好ましくは塩化ナトリゥムを含有しないかまたは塩化ナトリゥム濃度 0 . 2 M以下である。 特に好ましくは塩化ナトリウムを添加していない緩衝液を 使用する。 実施例 The present invention also provides a reagent kit for measuring HC II activity, comprising a reducing agent, a sulfated polysaccharide, thrombin, a thrombin substrate, a reaction terminator, and an HC II standard product. Reagents constituting the reagent kit of the present invention may be dissolved in purified water or a buffer so as to have a predetermined concentration in a reaction solution together with excipients, or diluted to a desired concentration at the time of use, according to a conventional method. It can be in the form of a concentrated solution or a lyophilized product. The reagent kit of the present invention may further contain a buffer for dilution. The buffer solution for dilution preferably has a low salt concentration. Specifically, the dilution buffer preferably does not contain sodium chloride or has a sodium chloride concentration of 0.25 M or less, and more preferably does not contain sodium chloride or has a sodium chloride concentration of 0 or less. . 2 M or less. Particularly preferably, a buffer to which sodium chloride is not added is used. Example
以下に実施例を挙げて本発明をさらに詳しく説明する。  Hereinafter, the present invention will be described in more detail with reference to Examples.
実施例 1 ：還元剤処理による AT- III の不活性化効果 Example 1: Inactivation effect of AT-III by treatment with reducing agent
(1) DTT処理  (1) DTT processing
10 mMの DTTを含むまたは DTTを含まない、 20 mM Tr i s— HC 1， pH 8の緩衝液で AT— III濃度を 5 UZm 1に調整し、 37 °Cで 30分間 の DTT処理を行った。 夾雑蛋白の影響を調べるために、 5%ヒト血清アルブミ ン （HSA) 存在下または非存在下で DTT処理を行った。  AT-III concentration was adjusted to 5 UZm1 with 20 mM Tris-HC1, pH 8 buffer containing 10 mM DTT or no DTT, and DTT treatment was performed at 37 ° C for 30 minutes. . To examine the effects of contaminating proteins, DTT treatment was performed in the presence or absence of 5% human serum albumin (HSA).
DTT処理した検体を、 DTTを含有しない検体希釈液 （0. 04M Tr i s , 0. 06 M エチレンジァミン四酢酸 （E DTA) , 0. 2 %H S A, 0. 1 mg/m 1 デルマタン硫酸， pH8. 56) で 100倍希釈し、 トロンビン 活性測定系に供した。  The DTT-treated sample was diluted with a sample diluent containing no DTT (0.04 M Tris, 0.06 M ethylenediaminetetraacetic acid (EDTA), 0.2% HSA, 0.1 mg / m 1 dermatan sulfate, pH 8. The solution was diluted 100-fold with 56) and used for a thrombin activity measurement system.
(2) トロンビン活性測定  (2) Thrombin activity measurement
希釈した検体溶液 50 1に、 0. 05%ゥシ血清アルブミ ン （B S A) ， 0. 05 %ポリエチレングリコール 6000 (PEG6000) を含有するヒ ト トロンビン溶液 （1. OU/m l) 100 1を添加し、 3 7でで 5分間静置し た。 ここに基質溶液 （S— 2238， 1. 52 mM) 100 ^ 1を添加し、 37 でで 5分間静置した。 2%クェン酸 1 000 μ 1を添加し反応を停止させた後、 405 nmにおける吸光度 （A 405 ) を測定した。 To the diluted sample solution 501, add a human thrombin solution (1. OU / ml) 1001 containing 0.05% ゥ serum albumin (BSA) and 0.05% polyethylene glycol 6000 (PEG6000). , 37 for 5 minutes. A substrate solution (S-2238, 1.52 mM) 100 ^ 1 was added thereto, and the mixture was allowed to stand at 37 for 5 minutes. After the reaction was stopped by adding 1,000 μl of 2% citric acid, the absorbance at 405 nm (A 405 ) was measured.
抗トロンビン活性は、 検体希釈液のみの場合のトロンビン活性に対する抑制率 であり、 下記式により求めた。 検体希釈液のみの場合の 一 検体のトロンビン活性  The antithrombin activity is the inhibition rate of the thrombin activity when only the sample diluent is used, and was determined by the following equation. Thrombin activity of one sample when only sample diluent is used
卜ロンビン活性  Trombin activity
X100(¾)  X100 (¾)
検体希釈液のみの場合のトロンビン活性  Thrombin activity with sample diluent only
DTT処理による抗トロンビン活性の抑制率を下記式により求めた。 結果を表 1に示す。 DTT処理しない場合の DTT処理した場合の The inhibition rate of antithrombin activity by DTT treatment was determined by the following equation. Table 1 shows the results. When DTT processing is not performed When DTT processing is performed
抗トロンビン活性 抗トロンビン活性 Antithrombin activity Antithrombin activity
100(¾)  100 (¾)
D T T処理しない場合の抗トロンビン活性  Antithrombin activity without DTT treatment
Figure imgf000009_0001
Figure imgf000009_0001
H S Aの有無にかかわらず、 AT— III を DTTで前処理すると、 無処理の場 合に比べて抗トロンビン活性は 8 7 %抑制された。 この結果から、 還元剤処理に より、 AT— III による影響を抑制できることが示された。 Pretreatment of AT-III with DTT, with or without HS A, reduced the antithrombin activity by 87% compared to no treatment. From these results, it was shown that the effect of AT-III can be suppressed by reducing agent treatment.
実施例 2 ： HCir活性測定 Example 2: HCir activity measurement
0丁丁溶液 （1001]^ DTT, 20 mM T r i s— HC 1， pH8) で H C II濃度を一定比 （1 : 2 : 4) に調整し、 3 7 、 3 0分間の DTT処理を行 つた。 DTT処理した反応液を、 DTTを含有しない検体希釈液 （0. 0 4 M T r i s , 0. 0 6 M EDTA, 0. 2 %H S A, 0. 1 mg/m 1 デルマ タン硫酸， p H 8. 5 6 ) で 1 0 0倍希釈し、 実施例 1と同様の方法で DTT処 理した場合の検体の抗トロンビン活性を測定した。 D T Tを含有しない検体希釈 液 （同上） で HCII標準品を希釈した。 実施例 1と同様の方法で各希釈標準品の 抗トロンビン活性を測定し、 検量線を作成した。 検量線を図 1に示す。 この検量 線に、 前記の DTT処理した検体の抗トロンビン活性を導入し、 DTT処理した 検体の HC II活性を算出した。 結果を表 2に示す。 0丁丁処理後の検体の11〇11 活性比が、 DTT処理をしていない検体の HCII濃度比と同じであった。 従って- DTT処理は HC II活性に影響を及ぼさないことが示された。 The HCII concentration was adjusted to a constant ratio (1: 2: 4) with 0-chome solution (1001] ^ DTT, 20 mM Tris-HC1, pH8), and DTT treatment was performed for 37 and 30 minutes. The DTT-treated reaction solution was added to a sample diluent containing no DTT (0.04 MT ris, 0.06 M EDTA, 0.2% HSA, 0.1 mg / m 1 dermatan sulfate, pH 8. The antithrombin activity of a sample when diluted 100-fold with 56) and subjected to DTT treatment in the same manner as in Example 1 was measured. The HCII standard was diluted with a sample diluent containing no DTT (same as above). The antithrombin activity of each diluted standard was measured in the same manner as in Example 1, and a calibration curve was prepared. Fig. 1 shows the calibration curve. The antithrombin activity of the DTT-treated sample was introduced into this calibration curve, and the HCII activity of the DTT-treated sample was calculated. Table 2 shows the results. 0 11〇11 of the sample after processing The activity ratio was the same as the HCII concentration ratio of the specimen without DTT treatment. Therefore, it was shown that -DTT treatment had no effect on HC II activity.
表 2  Table 2
Figure imgf000010_0001
実施例 3 ： HCII活性測定用試薬キッ ト
Figure imgf000010_0001
Example 3: Kit for measuring HCII activity
α) DTT溶液 （1 0mM DTT, 20 mM Tr i s—HC l, p H 8)α) DTT solution (10 mM DTT, 20 mM Tris—HCl, pH 8)
(2) 検体希釈液 （0. 04 M Tr i s， 0. 06 M EDTA, 0. 2 %H S A, 0. 1 mg/m 1 デルマタン硫酸， pH8. 56) (2) Sample diluent (0.04 M Tris, 0.06 M EDTA, 0.2% HS A, 0.1 mg / m 1 dermatan sulfate, pH 8.56)
(3) トロンビン溶液 （1. OUZml トロンビン， 0. 05%BSA, 0. 0 5%PEG6000)  (3) Thrombin solution (1. OUZml thrombin, 0.05% BSA, 0.05% PEG6000)
(4) 基質溶液 （S— 2238, 1. 52 mM)  (4) Substrate solution (S-2238, 1.52 mM)
(5) 反応停止剤 （2 %クェン酸）  (5) Reaction terminator (2% citrate)
(6) HCII標準品  (6) HCII standard product
以上の試薬からなる H C II活性測定用試薬キッ トを得た。  A reagent kit for measuring HC II activity comprising the above reagents was obtained.
産業上の利用可能性  Industrial applicability
本発明の測定方法によれば、 AT— III が混在する検体であっても、 AT— II I の影響を抑制し、 HCIIのみのトロンビン阻害活性を正確に測定することがで きる。 本発明の測定方法は、 簡便な操作により短時間で HCII活性の正確な測定 を行うことができる有用な方法である。 本出願は日本で出願された平成 10年特許願第 18993号を基礎としており- その内容は本明細書に全て包含されるものである。  According to the measurement method of the present invention, the effect of AT-III can be suppressed and the thrombin inhibitory activity of HCII alone can be accurately measured even for a sample containing AT-III. The measurement method of the present invention is a useful method that allows accurate measurement of HCII activity in a short time by a simple operation. This application is based on patent application No. 18993 filed in Japan, filed in Japan-the contents of which are incorporated in full herein.

Claims

請求の範囲 The scope of the claims
1 . へパリンコフアクター IIを含有する検体を還元剤で処理した後、 硫酸化多 糖の存在下でへパリンコファクタ一 IIをトロンビンと反応させ、 残存トロンビン 活性を測定することにより検体中のへパリンコフアクター IIのトロンビン阻害活 性を求めることを特徴とするへパリンコファクタ一 IIの活性測定方法。  1. After treating the sample containing heparin cofactor II with a reducing agent, react heparin cofactor-II with thrombin in the presence of sulfated polysaccharide, and measure the residual thrombin activity to determine the residual thrombin activity. A method for measuring the activity of heparin cofactor-1 II, which comprises determining the thrombin inhibitory activity of heparin cofactor II.
2 . 還元剤がジチオトレイ トールである請求項 1記載の方法。  2. The method according to claim 1, wherein the reducing agent is dithiothreitol.
3 . 還元剤による処理工程における反応液中の還元剤の濃度が 0 . 0 0 1 mM 〜1 0 O mMである請求項 1または 2記載の方法。  3. The method according to claim 1 or 2, wherein the concentration of the reducing agent in the reaction solution in the treatment step with the reducing agent is 0.001 mM to 10O mM.
4 . 硫酸化多糖がデルマタン硫酸である請求項 1〜 3のいずれか 1項に記載の 方法。  4. The method according to any one of claims 1 to 3, wherein the sulfated polysaccharide is dermatan sulfate.
5 . へパリンコファクタ一 IIとトロンビンの反応を低塩濃度の反応液中で行う 請求項 1〜 4のいずれか 1項に記載の方法。  5. The method according to any one of claims 1 to 4, wherein the reaction between heparin cofactor II and thrombin is performed in a reaction solution having a low salt concentration.
6 . へパリンコファクター IIと トロンビンの反応を、 塩化ナトリゥムを含有し ないかまたは塩化ナトリウム濃度 0 . 2 5 M以下の反応液中で行う請求項 1〜4 のいずれか 1項に記載の方法。  6. The method according to any one of claims 1 to 4, wherein the reaction between heparin cofactor II and thrombin is performed in a reaction solution containing no sodium chloride or a sodium chloride concentration of 0.25 M or less. .
7 . へパリンコファクタ一 IIとトロンビンの反応を、 塩化ナトリゥムを含有し ないかまたは塩化ナトリゥム濃度 0 . 2 M以下の反応液中で行う請求項 1〜4の いずれか 1項に記載の方法。  7. The method according to any one of claims 1 to 4, wherein the reaction between heparin cofactor II and thrombin is carried out in a reaction solution containing no sodium chloride or having a sodium chloride concentration of 0.2 M or less. .
8 . 還元剤、 硫酸化多糖、 トロンビン、 トロンビンの基質、 反応停止剤、 およ びへパリンコファクタ一II標準品からなるへパリンコファクタ一 Π活性測定用試 千ッ 卜 o  8. Reducing agent, sulfated polysaccharide, thrombin, thrombin substrate, reaction terminator, and heparin cofactor-II standard standard assay for heparin cofactor-II activity o
9 . 還元剤がジチォトレイ トールである請求項 8記載のへパリンコファクタ一 II活性測定用試薬キッ 卜。  9. The reagent kit for measuring heparin cofactor-II activity according to claim 8, wherein the reducing agent is dithiothreitol.
1 0 . 硫酸化多糖がデルマタン硫酸である請求項 8または 9記載のへパリンコ ファタター II活性測定用試薬キッ ト。  10. The reagent kit for measuring heparincophater II activity according to claim 8 or 9, wherein the sulfated polysaccharide is dermatan sulfate.
1 1 . さらに希釈用緩衝液を含む請求項 8〜1 0のいずれか 1項に記載のへパ リンコフアクター Π活性測定用試薬キッ ト。 11. The reagent kit for measuring the activity of heparin cofactor according to any one of claims 8 to 10, further comprising a buffer for dilution.
1 2 . 希釈用緩衝液が低塩濃度を有する緩衝液である請求項 1 1記載のへパリ ンコファクタ一 II活性測定用試薬キッ ト。 12. The reagent kit for measuring heparin cofactor-II activity according to claim 11, wherein the dilution buffer is a buffer having a low salt concentration.
1 3 . 希釈用緩衝液が塩化ナトリゥムを含有しないかまたは塩化ナトリゥム濃 度 0 . 2 5 M以下である請求項 1 1記載のへパリンコファクタ一 II活性測定用試 薬キッ ト。  13. The reagent kit for measuring heparin cofactor-II activity according to claim 11, wherein the dilution buffer does not contain sodium chloride or has a sodium chloride concentration of 0.25 M or less.
1 4 . 希釈用緩衝液が塩化ナトリゥムを含有しないかまたは塩化ナ卜リゥム濃 度 0 . 2 M以下である請求項 1 1記載のへパリンコファクタ一 II活性測定用試薬 ャッ 卜  14. The reagent kit for measuring heparin cofactor II activity according to claim 11, wherein the dilution buffer does not contain sodium chloride or has a sodium chloride concentration of 0.2 M or less.
PCT/JP1999/000291 1998-01-30 1999-01-22 Method for measuring heparin cofactor ii activity and reagent kit WO1999038997A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195608A1 (en) * 2000-10-03 2002-04-10 Roche Diagnostics GmbH Method, reagent, cartridge, and device for determining fibrinogen

Citations (2)

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Publication number Priority date Publication date Assignee Title
JPS60185163A (en) * 1984-03-02 1985-09-20 Dai Ichi Seiyaku Co Ltd Measurement of anti-thrombotic substance
JPH05503223A (en) * 1990-11-05 1993-06-03 バクスター、ダイアグノスチックス、インコーポレイテッド Anticoagulant concentration measurement method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60185163A (en) * 1984-03-02 1985-09-20 Dai Ichi Seiyaku Co Ltd Measurement of anti-thrombotic substance
JPH05503223A (en) * 1990-11-05 1993-06-03 バクスター、ダイアグノスチックス、インコーポレイテッド Anticoagulant concentration measurement method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195608A1 (en) * 2000-10-03 2002-04-10 Roche Diagnostics GmbH Method, reagent, cartridge, and device for determining fibrinogen
US6448024B1 (en) 2000-10-03 2002-09-10 Roche Diagnostics Corporation Method, reagent, cartridge, and device for determining fibrinogen

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