WO2017085947A1 - Procédé de mesure de l'activité de l'arginase, kit de détection de l'activité de l'arginase, et kit de détection de maladies associées à l'arginase - Google Patents

Procédé de mesure de l'activité de l'arginase, kit de détection de l'activité de l'arginase, et kit de détection de maladies associées à l'arginase Download PDF

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WO2017085947A1
WO2017085947A1 PCT/JP2016/061637 JP2016061637W WO2017085947A1 WO 2017085947 A1 WO2017085947 A1 WO 2017085947A1 JP 2016061637 W JP2016061637 W JP 2016061637W WO 2017085947 A1 WO2017085947 A1 WO 2017085947A1
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arginase
urea
solution
reaction
activity
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PCT/JP2016/061637
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English (en)
Japanese (ja)
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前野　恵美
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東京応化工業株式会社
田畑　泰彦
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Priority to EP16866421.7A priority Critical patent/EP3379248B1/fr
Priority to JP2017551939A priority patent/JP6884705B2/ja
Priority to PCT/JP2016/084208 priority patent/WO2017086421A1/fr
Publication of WO2017085947A1 publication Critical patent/WO2017085947A1/fr

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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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

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  • the present invention relates to a method for measuring arginase activity, an arginase activity detection kit, and an arginase-related disease detection kit.
  • This application claims priority based on Japanese Patent Application No. 2015-225939 filed in Japan on November 18, 2015 and Japanese Patent Application No. 2006-015903 filed in Japan on January 29, 2016. , The contents of which are incorporated herein.
  • Arginase is an enzyme located at the final stage of the urea cycle, and L-arginine is converted into L-ornithine and urea, and plays an important role in ammonium ion excretion from the body.
  • Arginase has two isoforms, type I and type II. Type I is mainly present in the liver and plays an important role in the urea cycle. Type II is present in organs including the kidney, and controls the concentrations of arginine and ornithine.
  • Arginase deficiency has been reported to cause serious symptoms such as neuropathy, dementia, and hyperammonemia, and measurement of arginase activity is essential for the study of urea metabolism.
  • arginase has been reported to have an anti-inflammatory action and an apoptosis (cell death) inhibitory effect of macrophages, and has recently been increasingly used as an index thereof.
  • Arginase activity was measured based on a series of enzymatic reactions in which arginase decomposes arginine into ornithine and urea, and urea further decomposes into ammonia and carbon dioxide by urease. 2) A production amount of urea, (3) a method by measurement of a production amount of ammonia, and the like (for example, see Patent Document 1).
  • Patent Document 1 the method for measuring the production amount of ornithine discloses a method for detecting ornithine by high performance liquid chromatography (HPLC), which requires a measuring instrument for HPLC, and further measures time. It takes.
  • HPLC high performance liquid chromatography
  • the method for measuring the amount of ammonia produced requires an enzyme reaction with urease, and thus the operation becomes complicated.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a highly sensitive and simple method for measuring arginase activity.
  • a sample containing arginase and a solution containing a divalent cation are added to a reaction vessel to activate arginase, and a sample containing activated arginase is added to a reaction vessel.
  • a second aspect of the present invention comprises arginine, a solution containing a divalent cation, and an acidic solution having a pH of 1.0 or more and 4.0 or less containing a urea detection reagent. It is a kit.
  • a third aspect of the present invention comprises an arginine, a solution containing a divalent cation, and an acidic solution having a pH of 1.0 or more and 4.0 or less containing a urea detection reagent. It is a kit.
  • arginase activity can be measured with high sensitivity and ease.
  • FIG. 3 is a diagram showing the measurement results of arginase activity in Example 1. It is a figure which shows the measurement result of the arginase activity in the comparative example 1. 3 is an image showing the measurement results of arginase activity in Example 2. 6 is an image showing a measurement result of arginase activity in Comparative Example 2.
  • the present invention adds a sample containing arginase and a solution containing a divalent cation to a reaction vessel to activate arginase, and a sample containing activated arginase.
  • a sample containing arginase and a solution containing a divalent cation to a reaction vessel to activate arginase, and a sample containing activated arginase.
  • Adding an arginine solution and performing an enzyme substrate reaction and adding an acidic solution containing a urea detection reagent to a pH of 1.0 or more and 4.0 or less to the sample subjected to the enzyme substrate reaction to deactivate the enzyme and And a step of simultaneously performing a detection reaction.
  • arginase activity can be measured with high sensitivity and ease.
  • FIG. 1 is a diagram showing a schematic configuration of a method for measuring arginase activity according to one embodiment.
  • ⁇ Arginase activation process First, a sample containing arginase is prepared and added to a reaction vessel together with a solution containing a divalent cation. Subsequently, in order to further increase the activity of arginase, incubation may be performed for about 10 minutes at a temperature at which the enzyme is not inactivated (for example, a temperature of 55 ° C. or higher and lower than 70 ° C.).
  • the sample containing arginase is not particularly limited.
  • body samples such as blood, saliva, tears, sweat, biological samples such as urine, suspensions of animal cells (eg, liver cells), animal cells A crushing liquid etc. are mentioned.
  • the sample containing arginase may be diluted with a buffer solution such as physiological saline or distilled water.
  • the divalent cation examples include Ca 2+ , Mg 2+ , Zn 2+ and Mn 2+ . Among these, Mn 2+ is preferable.
  • the solution containing the divalent cation is not particularly limited as long as the salt or hydrate containing the divalent cation is dissolved. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme activity of arginase can be increased by adding a solution containing a divalent cation to a sample containing arginase.
  • the concentration of the divalent cation in the solution may be, for example, from 1 mM to 100 mM, from 3 mM to 50 mM, and from 5 mM to 30 mM.
  • the reaction vessel is not particularly limited as long as it can measure arginase activity, and examples thereof include glass, metal, and plastic.
  • limiting in particular in the shape of reaction container For example, a glass slide shape, a microplate shape, a disk shape etc. are mentioned. Among these, since it is possible to measure a large number of samples, a microplate shape is preferable.
  • the microplate include those in which an arbitrary number of wells are arranged. Examples of the number of wells include 24, 96, 384, 1,536, etc., per plate.
  • the reaction vessel may constitute a microchannel device having a fine channel. The size of the reaction vessel may be in a range applicable to the apparatus to be used.
  • arginine solution is added as a substrate to a sample containing activated arginase, and an enzyme substrate reaction is performed.
  • the reaction temperature is preferably 20 ° C or higher and 45 ° C or lower, more preferably 30 ° C or higher and 40 ° C or lower, and most preferably 37 ° C or higher and 40 ° C or lower.
  • the reaction time is preferably from 30 minutes to 3 hours, particularly preferably 2 hours.
  • the pH during the reaction is preferably from 8.5 to 10.5, particularly preferably from 9.0 to 10.0.
  • the arginine solution is not particularly limited as long as it is a solution containing arginine (preferably L-arginine). It can be prepared by dissolving a salt or hydrate containing arginine in a solvent such as distilled water. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the urea detection reagent is not particularly limited as long as it can detect urea.
  • ⁇ -diketone p-dimethylaminobenzaldehyde, xanthohydrol and the like can be mentioned. Of these, ⁇ -diketone is preferred.
  • the ⁇ -diketone include ⁇ -isonitrosopropiophenone and diacetyl monooxime. Of these, ⁇ -isonitrosopropiophenone is preferable.
  • the acidic solution having a pH of 1.0 or more and 4.0 or less is not particularly limited as long as the pH can be adjusted within the above range.
  • the acid that can be used include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, salts and hydrates thereof, and the like.
  • a mixture of two or more of the above acids may be used. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme By mixing the urea detection reagent and the acidic solution having a pH of 1.0 or more and 4.0 or less in advance, the enzyme can be deactivated and urea can be detected in one operation. it can. Moreover, the addition amount of a solution can be decreased and the variation in the addition amount of a urea detection reagent can be reduced. Moreover, by making pH into the said range, an enzyme reaction and inactivation of an enzyme can fully be performed.
  • An acidic solution having a pH of 1.0 or more and 4.0 or less containing the urea detection reagent can be prepared, for example, by the following method.
  • a urea detection reagent, an acid, a salt or hydrate thereof, and a buffer solution such as physiological saline or distilled water are mixed if necessary. Furthermore, when the urea detection agent is relatively difficult to dissolve in the acidic solution, it is preferable to heat at the time of mixing. By heating, the urea detection reagent can be completely dissolved, and an acidic solution having a pH of 1.0 or more and 4.0 or less containing the urea detection reagent can be prepared.
  • the concentration of the urea detection reagent in the solution is preferably from 0.05% to 1.00%, more preferably from 0.08% to 0.70%, and more preferably from 0.10% to 0.00. More preferably, it is 65% or less.
  • the urea detection reaction may be promoted by incubating with heating after the addition of an acidic solution having a pH of 1.0 or more and 4.0 or less containing the urea detection reagent.
  • the heating temperature is preferably from room temperature to 100 ° C, particularly preferably from 90 ° C to 100 ° C. As heating time, it is 30 minutes or more, and 30 minutes or more and 12 hours or less are especially preferable. The heating temperature and the heating time can be appropriately adjusted according to the content of arginase. Moreover, by incubating with heating, the reaction by the urea detection reagent can be promoted, and detection can be performed earlier.
  • the detection of urea can be performed using a colorimetric method utilizing a reaction between urea and a urea detection reagent.
  • a colorimetric method utilizing a reaction between urea and a urea detection reagent.
  • the measuring device is not particularly limited as long as it can measure absorbance.
  • an absorptiometer spectrophotometer
  • a microplate reader etc. are mentioned.
  • the protein concentration in the sample may be measured to calibrate the measured value of arginase activity.
  • the protein concentration measurement method is not particularly limited, and examples thereof include an ultraviolet absorption method, a Bradford method (Coomassie blue method), a Lowry method (phenol reagent method), and a bicinchoninic acid method (BCA method).
  • the measurement method of the present embodiment can be efficiently measured using a small amount of sample and reagent. Further, conventionally, there has been a problem that the urea detection reagent discolors with time and the background becomes high. However, in the measurement method of the present embodiment, an acidic solution within the above pH range containing the urea detection reagent is previously stored. It is possible to make a measurement, and it is possible to measure in a state where the background is low with high sensitivity. Therefore, conventionally, measurement of several tens of samples is limited in one analysis, whereas the measurement method of the present embodiment can measure a large number of samples of several hundred samples or more.
  • the present invention provides an arginase activity detection kit comprising a solution containing arginine, a divalent cation, and an acidic solution having a pH of 1.0 or more and 4.0 or less containing a urea detection reagent. .
  • arginase activity can be detected with high sensitivity and ease.
  • arginine is not particularly limited in terms of origin as long as arginase can be decomposed as a substrate, but L-arginine is preferable.
  • Arginine may be a salt or a hydrate.
  • arginine may be a powder or a solution diluted with an appropriate solvent (for example, a buffer solution such as physiological saline, distilled water, etc.).
  • examples of the divalent cation include the same ones as described above, and among these, Mn 2+ is preferable.
  • the solution containing the divalent cation is not particularly limited as long as the salt or hydrate containing the divalent cation is dissolved. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme activity of arginase can be increased by adding a solution containing a divalent cation to a sample containing arginase.
  • the urea detection reagent is not particularly limited as long as urea can be detected.
  • the thing similar to what was mentioned above is mentioned.
  • ⁇ -isonitrosopropiophenone is preferable.
  • the acidic solution having a pH of 1.0 or more and 4.0 or less is not particularly limited as long as the pH can be adjusted within the above range.
  • the acid that can be used include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, salts and hydrates thereof, and the like.
  • a mixture of two or more of the above acids may be used. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme can be deactivated and the urea can be detected in a single operation, thereby improving the efficiency of the operation. be able to. Moreover, the addition amount of a solution can be decreased and the variation in the addition amount of a urea detection reagent can be reduced. Moreover, by making pH into the said range, an enzyme reaction and inactivation of an enzyme can fully be performed.
  • the present invention provides an arginase-related disease detection kit comprising arginine, a solution containing a divalent cation, and an acidic solution having a pH of 1.0 or more and 4.0 or less containing a urea detection reagent. .
  • an arginase-related disease can be detected with high sensitivity and ease.
  • examples of the “arginase-related disease” include heart disease, systemic hypertension, pulmonary hypertension, ischemia reperfusion injury, peripheral vascular disease, peripheral arterial disease, subarachnoid hemorrhage, erectile dysfunction, autoimmunity Encephalomyelitis, chronic renal failure, gastrointestinal dysfunction, gastric cancer, decreased hepatic blood flow, insufficient hepatic blood flow, cerebral vasospasm, or combinations thereof.
  • arginine is not particularly limited in terms of origin as long as arginase can be decomposed as a substrate, but L-arginine is preferable.
  • Arginine may be a salt or a hydrate.
  • arginine may be a powder or a solution diluted with an appropriate solvent (for example, a buffer solution such as physiological saline, distilled water, etc.).
  • examples of the divalent cation include those described above, and among these, Mn 2+ is preferable.
  • the solution containing the divalent cation is not particularly limited as long as the salt or hydrate containing the divalent cation is dissolved. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme activity of arginase can be increased by adding a solution containing a divalent cation to a sample containing arginase.
  • the urea detection reagent is not particularly limited as long as urea can be detected.
  • the thing similar to what was mentioned above is mentioned.
  • ⁇ -isonitrosopropiophenone is preferable.
  • the acidic solution having a pH of 1.0 or more and 4.0 or less is not particularly limited as long as the pH can be adjusted to the above range.
  • the acid that can be used include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, salts and hydrates thereof, and the like.
  • a mixture of two or more of the above acids may be used. Further, it may be diluted with a buffer solution such as physiological saline or distilled water.
  • the enzyme can be deactivated and the urea can be detected in a single operation, thereby improving the efficiency of the operation. be able to. Moreover, the addition amount of a solution can be decreased and the variation in the addition amount of a urea detection reagent can be reduced. Moreover, by making pH into the said range, an enzyme reaction and inactivation of an enzyme can fully be performed.
  • Example 1 Arginase activation step 20 ⁇ L of a mixed solution of 50 mM Tris-HCl (pH 7.5) and 10 mM MnCl 2 was dispensed into a microplate with a multipipetter. Subsequently, animal cells were disrupted using a mixed solution of 0.1% Triton X100 and Proteinase Inhibitor mixture to prepare a cell disruption solution as a sample containing arginase. Subsequently, 20 ⁇ L of the prepared cell disruption solution was added to the microplate by a multipipette. Subsequently, arginase was activated by incubation at 56 ° C. for 10 minutes.
  • the negative control (Negative control) was obtained by performing an enzyme substrate reaction and urea detection reaction using a cell disruption solution to which an arginase activity inhibitor was added, and a sample (Sample). ) Shows an enzyme substrate reaction and urea detection reaction using a cell disruption solution to which an arginase activity promoting factor and an arginase activity inhibiting factor were added.
  • the negative control (Negative control) was obtained by performing an enzyme substrate reaction and urea detection reaction using a cell disruption solution to which an arginase activity inhibitor was added, and a sample (Sample). ) Shows an enzyme substrate reaction and urea detection reaction using a cell disruption solution to which an arginase activity promoting factor and an arginase activity inhibiting factor were added.
  • Example 2 and 3 the measured value of arginine activity of the sample was twice as high in Comparative Example 1 as compared to the measured value of arginine activity in the negative control, and was 5 times higher in Example 1. Further, in Example 1, no significant difference was obtained between the measured value of arginine activity in the control and the measured value of arginine activity in the sample, whereas in Example 1, there was no significant difference between any conditions. A difference was obtained. In the method for measuring arginase activity described in Comparative Example 1, it is presumed that the urea detection reagent changed color over time and the background increased. From the above, it was confirmed that the method for measuring arginase activity described in Example 1 has a low background and can measure arginase activity with high sensitivity.
  • Example 2 (1) Preparation of urea solution Using urea (manufactured by Nacalai Tesque), urea solutions were prepared so as to be 0, 5, 10, 20, 40, 80, and 160 ⁇ g / mL.
  • “mean” is a value obtained by calculating an average value of 8 wells at each of urea concentrations of 0, 5, 10, 20, 40, 80, and 160 ⁇ g / mL, and then dividing the average value by a standard deviation (average (Value / standard deviation) is calculated, and a value obtained by averaging the calculated seven average values / standard deviation is shown. “SD” indicates the standard deviation of the seven average values / standard deviation. Moreover, the image which image
  • Example 2 tended to have a smaller mean value compared to Comparative Example 2.
  • Comparative Example 2 the mean value varied throughout the heating time. Further, Example 2 tended to have a smaller SD value than Comparative Example 2 except for the sample heated for 8 hours. From the above, it has been clarified that the measurement method of the present invention can reduce variations in measured values.
  • Example 2 in Example 2, almost no discoloration of the urea detection reagent was observed in 8 hours, whereas in Comparative Example 2, discoloration of the urea detection reagent was confirmed in 8 hours. .
  • the ethanol solvent contained in the urea detection reagent evaporates during the dispensing operation, and the concentration of ⁇ -isonitrosopropiophenone increases, whereas in Example 2, It is presumed that the acidic solution containing the urea detection reagent is dispensed after being mixed in advance, and the ethanol solvent can be advanced to the next step without evaporating. Therefore, it was clarified that Comparative Example 2 which is a conventional measurement method has a high background, whereas the measurement method of the present invention has a low background and can detect arginase activity more accurately.
  • arginase activity can be measured with high sensitivity and ease.

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Abstract

Un procédé de mesure de l'activité de l'arginase selon la présente invention est caractérisé en ce qu'il comprend : une étape consistant à activer l'arginase en ajoutant un échantillon contenant de l'arginase et une solution contenant un cation divalent dans une cuve de réaction ; une étape consistant à mettre en œuvre une réaction enzyme-substrat en ajoutant une solution d'arginine à l'échantillon contenant l'arginase activée ; et une étape consistant à mettre en œuvre simultanément la désactivation de l'enzyme ainsi que la réaction de détection de l'urée en ajoutant à l'échantillon, sur lequel a été mise en œuvre une réaction enzyme-substrat, une solution acide ayant un pH de 1,0 à 4,0 et qui contient un réactif de détection d'urée.
PCT/JP2016/061637 2015-11-18 2016-03-31 Procédé de mesure de l'activité de l'arginase, kit de détection de l'activité de l'arginase, et kit de détection de maladies associées à l'arginase WO2017085947A1 (fr)

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Application Number Priority Date Filing Date Title
EP16866421.7A EP3379248B1 (fr) 2015-11-18 2016-11-18 Procédé de mesure de l'activité de l'arginase, kit de détection de l'activité de l'arginase, kit de détection de maladies associées à l'arginase, et procédé de criblage à la recherche d'agents actifs ou d'inhibiteurs de l'arginase
JP2017551939A JP6884705B2 (ja) 2015-11-18 2016-11-18 アルギナーゼ活性の測定方法、アルギナーゼ活性の検出キット、アルギナーゼ関連疾患検出キット、及びアルギナーゼの阻害剤又は活性剤のスクリーニング方法
PCT/JP2016/084208 WO2017086421A1 (fr) 2015-11-18 2016-11-18 Procédé de mesure de l'activité de l'arginase, kit de détection de l'activité de l'arginase, kit de détection de maladies associées à l'arginase, et procédé de criblage à la recherche d'agents actifs ou d'inhibiteurs de l'arginase

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JP2015-225939 2015-11-18
JP2015225939 2015-11-18
JP2016015903 2016-01-29
JP2016-015903 2016-01-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010500973A (ja) * 2006-07-21 2010-01-14 マース インコーポレーテッド アルギナーゼ濃度／活性の改善
JP2015516397A (ja) * 2012-04-18 2015-06-11 マーズ インコーポレイテッド アルギナーゼ阻害剤としての環拘束性類似体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010500973A (ja) * 2006-07-21 2010-01-14 マース インコーポレーテッド アルギナーゼ濃度／活性の改善
JP2015516397A (ja) * 2012-04-18 2015-06-11 マーズ インコーポレイテッド アルギナーゼ阻害剤としての環拘束性類似体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALMY, J ET AL.: "Urea Analysis for Wines", J. AGRIC. FOOD CHEM., vol. 37, 1989, pages 968 - 970, XP055488045 *

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