JP2007028937A - Reagent for measuring activity of creatine kinase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent for measuring activity of CK (creatine kinase) preventing deterioration of SH compound in the reagent solution and preventing increase of optical absorbance of the reagent itself. <P>SOLUTION: The reagent for measuring activity of CK comprises cyclohexyldiamine tetraacetic acid, and the compound having a SH group. The invention relates to a reagent kit for measuring activity of CK comprising a first reagent comprising the cyclohexyldiamine tetraacetic acid, glucose-6-phosphate dehydrogenase, NAD(P), hexokinase or glucokinase, and ADP, and a second reagent comprising creatine phosphate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reagent for measuring the activity of creatine kinase (CK) in a sample and a method for stabilizing a compound having an SH group (hereinafter referred to as an SH compound).

  CK is a dimeric phosphorylase consisting of two subunits. There are two types of CK subunits, B type (brain type) and M type (muscle type). CK has three types of isozymes (CK-MM, CK-MB, and CK-BB) by combining two types of subunits. CK-MM is abundant in skeletal muscle and CK-MB is in myocardium. Many CK-BBs are contained in the brain. Since CK isozymes present at the disease-causing site deviate into the blood due to diseases such as myocardial infarction and muscular dystrophy, the activity value of CK contained in samples such as serum and plasma in clinical tests is important in diagnosing the above diseases Index.

  Since CK is quickly inactivated in blood, an activity measurement is performed after activating CK by adding an SH compound to a sample collected from a living body. Therefore, the reagent for measuring CK activity contains SH compounds such as N-acetyl-L-cysteine (hereinafter referred to as NAC) and thioglycerol.

  The SH compound is an unstable substance, and the SH compound is deteriorated by the gradual oxidation of the SH group during storage of the reagent. For this reason, it is necessary to make the reagent contain a substance that stabilizes the SH compound. A chelating agent is used as a substance that stabilizes the SH compound. As a chelating agent for stabilizing the SH compound, diethylenetriaminepentaacetic acid (DTPA) is known (Patent Document 1). However, although DTPA can stabilize the SH compound, the absorbance of the reagent itself increases while the reagent is stored. For this reason, the absorbance immediately after mixing the reagent and the sample (initial absorbance) also increases, and the measurement accuracy may decrease.

Japanese Patent Laid-Open No. 11-032798

  An object of the present invention is to provide a reagent for measuring CK activity containing a chelating agent that can prevent the deterioration of the SH compound in the reagent solution and suppress the increase in absorbance of the reagent itself, and such a chelating agent. To provide a method for stabilizing SH compounds.

  The present invention provides a reagent for measuring CK activity containing cyclohexyldiaminetetraacetic acid (hereinafter referred to as CyDTA) and an SH compound.

  The present invention also provides a method for stabilizing an SH compound, characterized by coexisting CyDTA and an SH compound.

  ADVANTAGE OF THE INVENTION According to this invention, the reagent for CK activity measurement excellent in storage stability containing the chelating agent which can stabilize SH compound and can suppress the raise of the light absorbency of the reagent itself can be provided. Moreover, the method of stabilizing an SH compound using such a chelating agent can be provided.

  The reagent for measuring CK activity of this embodiment contains CyDTA and an SH compound. CyDTA is a white powder, and its chemical name is trans-1,2-Diaminocyclohexane-N, N, N ′, N′-tetraacetic acid and has a chelating action. The concentration of CyDTA in the reagent is not particularly limited as long as it is a concentration capable of stabilizing the SH compound. As used herein, “CyDTA” includes a salt of CyDTA. Examples of the salt of CyDTA include a potassium salt, a sodium salt, and a magnesium salt of CyDTA.

  The SH compound is not particularly limited as long as CK in the sample can be activated. For example, N-acetyl-L-cysteine (NAC), N-guanyl-L-cysteine, cysteamine, dithiothreitol (DTT), cysteine, Glutathione, mercaptosuccinic acid, thioglucose, dithioerythritol, mercaptoacetic acid, 2-mercaptoethanol, 2-aminoethylisothiouronium bromide, 2-mercaptoethanesulfonic acid, thioglycerol and the like can be used. These SH compounds may be used in combination of two or more, but are preferably used alone. The concentration (final concentration) of the SH compound in the reaction mixture in which the sample and the reagent are mixed is 0.1 to 200 mM, preferably 10 to 100 mM.

  Although it does not specifically limit as pH in the solution state of the reagent for CK activity measurement, It is preferable that it is 5.0-10.0. In order to maintain the pH, it is preferable to include a buffer in the reagent. Examples of the buffer include Tris-HCl buffer, imidazole-acetate buffer, phosphate buffer, citrate buffer, malate buffer, oxalate buffer, phthalate buffer, glycine buffer, acetate buffer. Succinic acid buffer, boric acid buffer, carbonic acid buffer, Good buffer, and the like can be used.

  The reagent for measuring CK activity includes glucose-6-phosphate dehydrogenase (G6PDH), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), hexokinase (HK) or glucokinase (GK). ), Glucose, adenosine diphosphate (ADP) and creatine phosphate.

The reagent for measuring CK activity may be in a state in which each component is freeze-dried or in a solution state in which it is dissolved in a solvent.
The G6PDH-containing reagent is preferably composed of two reagents, a first reagent and a second reagent. In this case, the first reagent and / or the second reagent may be lyophilized, or both reagents may be in solution. CyDTA and SH compounds may be contained in either the first reagent or the second reagent, and may be contained in both, but are preferably contained in the first reagent.

  When the reagent for measuring CK activity comprises a first reagent and a second reagent, the first reagent includes G6PDH, CyDTA, NAD or NADP, hexokinase (HK) or glucokinase (GK), glucose, adenosine diphosphate (ADP) It is preferable that magnesium ion and SH compound are contained, and creatine phosphate is contained in the second reagent.

  The origin of G6PDH, HK and GK is not particularly limited, and G6PDH, HK and GK may be those derived from bacteria, yeast, animals and plants, or those generated using genetic recombination techniques. The final concentration of G6PDH is 0.5 to 40 U / ml, preferably 1 to 10 U / ml. The final concentration of HK or GK is 0.5 to 20 U / ml, preferably 1 to 6 U / ml.

The measurement target of the reagent for measuring CK activity of this embodiment is any one of the activities of CK, CK-MM, CK-MB, and CK-BB included in the sample. In general, in a clinical test, the activity of all CK or CK-MB in a sample is measured. By containing an antibody that specifically binds to the M-type subunit of CK (hereinafter referred to as anti-CK-M antibody) in the reagent for measuring CK activity, the activity of CK-MB in the sample can be measured. (Wurzburg et al., 1977, J. Clin. Chem. Clin. Biochem., 15: 131-135). The anti-CK-M antibody may be a polyclonal antibody or a monoclonal antibody as long as it is an antibody that specifically recognizes the M-type subunit, and these may be used in combination. Antibody fragments and derivatives thereof can also be used. Specific examples of antibody fragments and derivatives thereof include Fab, Fab ′, F (ab) ₂ and sFv fragments (Blazar et al., 1997, J. Immunol., 159: 5821-5833 and Bird et al. , 1988, Science, 242: 423-426). The subclass of the antibody is not limited to IgG, and may be IgM.

  As a sample to be used for measurement, for example, serum, plasma, blood, spinal fluid, urine, semen and the like can be used, but it is preferable to use plasma or serum.

Hereinafter, the reaction system used for CK activity measurement will be described.
A reaction system as shown in FIG. 1 is constructed by adding a reagent for measuring CK activity containing the above components to a sample containing CK. In FIG. 1, CK activated by an SH compound catalyzes a reaction for producing creatine and ATP from creatine phosphate and ADP (reaction 1). HK or GK contained in the reagent produces glucose-6-phosphate (G6P) and ADP from glucose contained in the reagent and ATP produced in Reaction 1 (Reaction 2). Further, G6PDH contained in the reagent generates 6-phosphoglucono-δ-lactone and NADH or NADPH from G6P and NAD or NADP generated in Reaction 2 (Reaction 3). When NADH or NADPH is generated, the absorbance of the mixed solution of the sample and the reagent near the wavelength of 340 nm increases. By monitoring this increase in absorbance, the activity of CK in the sample can be measured.

  As described above, since the SH compound in the reagent gradually deteriorates during storage of the reagent, it must be stabilized. Usually, a chelating agent is added to the reagent for the purpose of stabilizing the SH compound. Although DTPA, which is a kind of such a chelating agent, can prevent the deterioration of the SH compound even when the reagent is stored for a long period of time, the absorbance of the reagent itself increases during storage of the reagent. In addition, EDTA, which is recommended as a chelating agent that stabilizes SH compounds by the recommended method by the Japanese Society for Clinical Chemistry, does not increase the absorbance of the reagent itself even if the reagent is stored for a long period of time, but sufficiently suppresses deterioration of the SH compound. I can't. Furthermore, after storing a reagent containing EDTA for a long period of time, when a CK activity measurement is performed on a sample having a high CK activity using this reagent, the SH compound in the reagent is deteriorated during storage, so that it is included in the sample. CK may not be activated sufficiently and may not be measured accurately.

  By causing CyDTA and SH compound to coexist in the reagent for measuring CK activity, it is possible to suppress degradation of SH groups due to long-term storage of the reagent. CyDTA can suppress an increase in absorbance of the reagent itself. Furthermore, according to the reagent for measuring CK activity containing CyDTA, even when the activity measurement is performed using a sample having a high CK activity, the deterioration of the SH compound can be suppressed, so that the CK activity can be accurately measured. Is possible.

  It is preferable to contain magnesium ions in the reagent for measuring CK activity. Magnesium ions can be contained by adding a magnesium salt to the reagent. As the magnesium salt, magnesium acetate, magnesium sulfate, magnesium chloride and the like can be used.

  A preservative or a compound having a surfactant activity may be added to the reagent. As the preservative, for example, sodium azide can be used. As the compound having a surface activity, for example, a nonionic surfactant, a cationic surfactant, a zwitterionic surfactant, albumin and the like can be used. Specifically, Tritons (Union Carbide Chemicals and Plastics Co. ), Emulgens (registered trademark of Kao Corporation), BSA, etc. can be used.

  The sample may contain adenylate kinase. Adenylate kinase is particularly abundant in hemolyzed samples and adversely affects CK activity measurement. In order to avoid this adverse effect, it is preferable to add an inhibitor that inhibits the action of adenylate kinase to the reagent. The type of inhibitor is not particularly limited as long as it inhibits the action of adenylate kinase. For example, adenosine monophosphate (AMP), P1P5 diadenosine-5′-pentaphosphate (AP5A), or the like is used. it can.

  Furthermore, the adverse effect of adenylate kinase can be avoided by measuring the activity by the double kinetic method. In the double kinetic method, first, the activity of adenylate kinase is measured, and then creatine phosphate is added to initiate the enzymatic reaction with CK to activate the kinase activity contained in the sample (CK activity and adenylate kinase activity). Activity). The difference between these measurement results is the CK activity value.

Example 1
The following substances were dissolved in purified water to the following concentrations.
Imidazole 125 mM
Magnesium acetate 12.5 mM
ADP 2.5 mM
AMP 6.25 mM
AP5A 12.5μM
Glucose 25 mM
NADP 2.5 mM
Thioglycerol 44 mM
G6PDH 1875U / L
Hexokinase 3750U / L
The pH of the solution was adjusted to 6.6.

  CyDTA was added to the above composition to prepare three types of CK activity measurement reagents having different CyDTA concentrations. The CyDTA concentrations of these reagents were 1 mM, 2 mM and 4 mM, respectively. These reagents were subjected to a temperature load at 37 ° C. for 5 days, and then the residual amount of SH groups in the reagents was quantified.

  The reagent for measuring CK activity consists of the above-mentioned reagent (first reagent) and a reagent containing creatine phosphate (second reagent). In this example, the above-mentioned reagent was subjected to a temperature load to evaluate the stability of thioglycerol. Therefore, the second reagent is not used.

  The SH group was quantified using DTNB (5,5′-dithiobis (2-nitrobenzoic acid)). When DTNB is added to the reagent, when an SH compound is present, an amount of disulfide bond corresponding to the amount of SH group of the SH compound is broken to produce 5-Mercapto-2-nitrobenzoic acid. When 5-Mercapto-2-nitrobenzoic acid is produced, the absorbance at a wavelength of 412 nm increases. By measuring this, the SH group in the reagent was quantified.

(Comparative Examples 1-6)
In Comparative Example 1, the SH group of the reagent stored at −80 ° C. was quantified. Here, no chelating agent is added and no temperature load is applied.
In Comparative Example 2, the amount of SH group remaining in thioglycerol was quantified in the same manner as in Example 1 except that no chelating agent was added.
In Comparative Example 3, the amount of SH group remaining in thioglycerol was quantified in the same manner as in Example 1 except that DTPA was added to the above composition instead of CyDTA, and three types of reagents having different DTPA concentrations were prepared. . The DTPA concentration of each reagent was 1 mM, 2 mM and 4 mM.
In Comparative Example 4, the remaining amount of SH groups in thioglycerol was determined in the same manner as in Example 1 except that glycolethylenediaminetetraacetic acid (GEDTA) was added instead of CyDTA, and three types of reagents having different GEDTA concentrations were prepared. Was done. The GEDTA concentration of each reagent was 1 mM, 2 mM and 4 mM.
In Comparative Example 5, the amount of residual SH groups in thioglycerol was quantified in the same manner as in Example 1 except that ethylenediaminetetraacetic acid (EDTA) was added instead of CyDTA, and three types of reagents with different EDTA concentrations were prepared. I did it. The EDTA concentration of each reagent was 1 mM, 2 mM and 4 mM.
In Comparative Example 6, the remaining amount of SH groups in thioglycerol was quantified in the same manner as in Example 1 except that EDTA-OH was added instead of CyDTA, and three types of reagents with different EDTA-OH concentrations were prepared. It was. The EDTA-OH concentration of each reagent was 1 mM, 2 mM and 4 mM.
In Comparative Example 7, quantification of the residual amount of SH groups in thioglycerol was performed in the same manner as in Example 1 except that ethylenediaminediacetic acid (EDDA) was added instead of CyDTA, and three types of reagents with different EDDA concentrations were prepared. I did it. The EDDA concentration of each reagent was 1 mM, 2 mM and 4 mM.

  The measurement results of Example 1 and Comparative Examples 1 to 7 are shown in Table 1 below. In Table 1, the remaining amount of thioglycerol is shown as a percentage. These values are values for the measurement results of Comparative Example 1.

  From the measurement results of Example 1 and Comparative Examples 2 to 7, the reagent containing CyDTA (Example 1) and the reagent containing DTPA (Comparative Example 3) were converted into reagents containing no chelating agent and other chelating agents. In comparison, the residual amount of SH groups after temperature loading was large. That is, it was found that the storage stability of thioglycerol can be improved by adding DTPA or CyDTA to the reagent.

(Example 2)
The following substances were dissolved in purified water to the following concentrations to prepare first and second reagents for measuring CK activity, and the initial absorbance was measured using them.

<First reagent> pH 6.6
Imidazole 125 mM
Magnesium acetate 12.5 mM
ADP 2.5 mM
AMP 6.25 mM
AP5A 12.5μM
Glucose 30 mM
NADP 2.5 mM
NAC 44 mM
G6PDH 1875U / L
Hexokinase 3750U / L
CyDTA 2mM
<Second reagent> pH 9.0
Creatine phosphate 150 mM

  Using a Hitachi 7170S automatic analyzer, 180 μl of the first reagent, 45 μl of the second reagent, and 5.6 μl of the sample which is a physiological saline not containing CK were mixed, and the absorbance at 340 nm was measured.

  The first reagent stored at 4 ° C. for 1 week, the first reagent stored at 4 ° C. for 2 weeks, the first reagent stored at 4 ° C. for 3 weeks, and the first reagent stored at 4 ° C. for 4 weeks, respectively. The initial absorbance was measured in the same manner as described above.

(Comparative Examples 8 to 13)
The initial absorbance was measured by adding another type of chelating agent instead of CyDTA in the composition of the first reagent.
In Comparative Example 8, the initial absorbance was measured in the same manner as in Example 2 except that 2 mM of DTPA was added instead of CyDTA.
In Comparative Example 9, the initial absorbance was measured in the same manner as in Example 2 except that 2 mM of GEDTA was added instead of CyDTA.
In Comparative Example 10, the initial absorbance was measured in the same manner as in Example 1 except that 2 mM of EDTA was added instead of CyDTA.
In Comparative Example 11, the initial absorbance was measured in the same manner as in Example 1 except that 2 mM of EDTA-OH was added instead of CyDTA.
In Comparative Example 12, the initial absorbance was measured in the same manner as in Example 1 except that 2 mM of EDDA was added instead of CyDTA.
In Comparative Example 13, the initial absorbance was measured in the same manner as in Example 1 except that 2 mM nitrilotriacetic acid (NTA) was added instead of CyDTA.

  The measurement results of Example 2 and Comparative Examples 8 to 13 are shown in Table 2 below. In addition, the value in Table 2 is a value obtained by multiplying the measured absorbance by 10,000. In the column of the increase rate, the percentage of the increase in the initial absorbance after storage for 4 weeks compared with the initial absorbance immediately after the reagent preparation was shown as a percentage.

  From the measurement results of Example 2 and Comparative Examples 8 to 13, a reagent to which CyDTA was added (Example 2), a reagent to which EDTA was added (Comparative Example 10), and a reagent to which EDTA-OH was added (Comparative Example 11) Compared to reagents containing other chelating agents, the rate of increase in initial absorbance due to long-term storage was low. That is, it was found that CyDTA, EDTA and EDTA-OH can suppress an increase in absorbance of the reagent itself.

(Example 3)
Using the first reagent and the second reagent prepared in Example 2, the CK activity of a sample containing about 2000 U / l of CK was measured.

  Using a Hitachi 7170S automatic analyzer, 180 μl of the first reagent, 45 μl of the second reagent, and 5.6 μl of the sample were mixed and the absorbance at 340 nm was measured, and the activity value of CK contained in the sample was calculated.

  The CK activity value was calculated in the same manner as described above using the first reagent stored at 4 ° C. for 2 weeks and the first reagent stored at 4 ° C. for 4 weeks.

(Comparative Examples 14-19)
In Comparative Example 14, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 8 was used.
In Comparative Example 15, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 9 was used.
In Comparative Example 16, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 10 was used.
In Comparative Example 17, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 11 was used.
In Comparative Example 18, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 12 was used.
In Comparative Example 19, the CK activity value was calculated in the same manner as in Example 3 except that the first reagent prepared in Comparative Example 13 was used.

  The measurement results of Example 3 and Comparative Examples 14 to 19 are shown in Table 3 below. In the column of the reduction rate, the percentage of the value after storage for 4 weeks compared with the CK activity value immediately after the reagent preparation was shown as a percentage.

  From the measurement results of Example 3 and Comparative Examples 14 to 19, the reagent to which CyDTA was added (Example 3) and the reagent to which DTPA was added (Comparative Example 14) were stored for 4 weeks as compared with reagents containing other chelating agents. The decrease rate of the measured value after the measurement was small. That is, it was found that by using a reagent to which CyDTA or DTPA was added, CK activity could be accurately measured even when the reagent was stored for a long period of time.

  From Examples 1, 2, 3 and Comparative Examples 1-18, the reagent containing CyDTA has excellent storage stability of the SH compound, and the absorbance of the reagent itself does not increase greatly even after storage for a long period of time. According to the reagent, it was found that the activity can be accurately measured even when a sample having a high CK activity is used.

It is the schematic diagram which showed the reaction system of CK activity measurement.

Claims (6)

  A reagent for measuring creatine kinase activity, comprising cyclohexyldiaminetetraacetic acid and a compound having an SH group.   The compound having an SH group is N-acetyl-L-cysteine (NAC), N-guanyl-L-cysteine, cysteamine, dithiothreitol (DTT), cysteine, glutathione, mercaptosuccinic acid, thioglucose, dithioerythritol, The reagent for measuring creatine kinase activity according to claim 1, which is at least one selected from the group consisting of mercaptoacetic acid, 2-mercaptoethanol, 2-aminoethylisothiouronium bromide, 2-mercaptoethanesulfonic acid and thioglycerol. .   Glucose-6-phosphate dehydrogenase (G6PDH), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), hexokinase or glucokinase, glucose, adenosine diphosphate (ADP) and creatine phosphorus The reagent for measuring creatine kinase activity according to claim 1 or 2, further comprising at least one compound selected from the group consisting of acids.   The reagent for measuring creatine kinase activity according to any one of claims 1 to 3, further comprising an antibody that specifically binds to the M-type subunit of creatine kinase.   Cyclohexyldiaminetetraacetic acid (CyDTA), glucose-6-phosphate dehydrogenase (G6PDH), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), hexokinase or glucokinase, glucose and adenosine A reagent kit for measuring creatine kinase activity, comprising a first reagent containing phosphoric acid (ADP) and a second reagent containing creatine phosphate. A method for stabilizing a compound having an SH group, the method comprising stabilizing a compound having an SH group, wherein cyclohexyldiaminetetraacetic acid and a compound having an SH group coexist.

Images