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WO1999038997A1 - Procede de mesure de l'activite du cofacteur ii de l'heparine et boite de reactifs y relative - Google Patents

Procede de mesure de l'activite du cofacteur ii de l'heparine et boite de reactifs y relative 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
Authority
WO
WIPO (PCT)
Prior art keywords
activity
thrombin
measuring
heparin cofactor
hcii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1999/000291
Other languages
English (en)
Japanese (ja)
Inventor
Takashi Goto
Naomi Asahara
Akimasa Omizu
Yahiro Uemura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsubishi Tanabe Pharma Corp
Original Assignee
Yoshitomi Pharmaceutical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yoshitomi Pharmaceutical Industries Ltd filed Critical Yoshitomi Pharmaceutical Industries Ltd
Priority to AU19836/99A priority Critical patent/AU1983699A/en
Publication of WO1999038997A1 publication Critical patent/WO1999038997A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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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  • General Physics & Mathematics (AREA)
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Abstract

L'invention concerne un procédé pour la mesure, à haute précision, de l'activité du cofacteur II de l'héparine (HC II). Ce procédé consiste à traiter un échantillon contenant HC II par un agent réducteur, à faire réagir HC II avec la thrombine, en présence d'un polysaccharide sulfaté, et à mesurer l'activité résiduelle de la thrombine de façon à déterminer l'activité inhibitrice de la thrombine de HC II dans l'échantillon. L'invention concerne en outre un ensemble de réactifs utilisés pour la mesure de l'activité de HC II, comprenant un agent réducteur, un polysaccharide sulfaté, la thrombine, un substrat de thrombine, un agent d'arrêt de la réaction et un étalon HC II: Dans le procédé précité, l'antithrombine III (AT-III) porteuse de liaison S-S est inactivée sélectivement par le traitement par l'agent réducteur. De cette façon, l'activité inhibitrice de la thrombine de HC II seul peut être mesurée avec précision, même dans le cas d'un échantillon contaminé par AT-III.
PCT/JP1999/000291 1998-01-30 1999-01-22 Procede de mesure de l'activite du cofacteur ii de l'heparine et boite de reactifs y relative Ceased WO1999038997A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19836/99A AU1983699A (en) 1998-01-30 1999-01-22 Method for measuring heparin cofactor ii activity and reagent kit

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP1899398 1998-01-30
JP10/18993 1998-01-30

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WO1999038997A1 true WO1999038997A1 (fr) 1999-08-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195608A1 (fr) * 2000-10-03 2002-04-10 Roche Diagnostics GmbH Procédé, réactif, cartouche et dispositif d'essai destinés a détérminer fibrinogène

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60185163A (ja) * 1984-03-02 1985-09-20 Dai Ichi Seiyaku Co Ltd 抗トロンビン物質の測定法
JPH05503223A (ja) * 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 抗凝固剤濃度測定方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60185163A (ja) * 1984-03-02 1985-09-20 Dai Ichi Seiyaku Co Ltd 抗トロンビン物質の測定法
JPH05503223A (ja) * 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 抗凝固剤濃度測定方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195608A1 (fr) * 2000-10-03 2002-04-10 Roche Diagnostics GmbH Procédé, réactif, cartouche et dispositif d'essai destinés a détérminer fibrinogène
US6448024B1 (en) 2000-10-03 2002-09-10 Roche Diagnostics Corporation Method, reagent, cartridge, and device for determining fibrinogen

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AU1983699A (en) 1999-08-16

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