JP3161316B2 - Liquid reagent for measuring creatine kinase activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a creatine kinase (hereinafter abbreviated as CK) activator for a liquid reagent and a method for measuring CK activity in a body fluid such as serum using the activator. It relates to a liquid reagent.

[0002]

2. Description of the Related Art In vivo, CK is most frequently present in skeletal muscles, followed by brain and myocardium, and also in smooth muscle and nervous system. not exist. In 1959, the rise in serum of patients with advanced muscular dystrophy was followed by clinical application, and the measurement of CK activity in the field of clinical tests is currently used for muscle diseases,
It is one of the important items that are routinely performed for diagnosis of heart disease and the like.

On the other hand, since CK is rapidly inactivated in serum, when CK activity is measured, an activating agent is added to an activity measuring reagent, and CK is activated by the activating agent. It is common to make measurements later. Examples of CK activators include so-called SH group-containing compounds (hereinafter abbreviated as SH compounds) such as N-acetylcysteine (hereinafter abbreviated as NAC), dithiothreitol (hereinafter abbreviated as DTT), glutathione and the like. Is generally used, but as the CK activator, various SH
Compounds have been investigated. For example, 1968 Hess
Is cysteine, 1972 Warren is DTT,
In 1976 Szats used NAC as a CK activator, respectively [Clinical Chemistry, Vol. 19, No. 2, 184
To 208 (1990), and Clin. Chem., Vol. 23, 1569 to 1575.
(1977)]. Further, in 1977, Morin announced that thioglycerol (hereinafter abbreviated as TG) and mercaptoethanol were suitable as CK activators [Clin. Chem., Vol. 23, 1569-1575 (1977)]. . However, in the literature, only the examination of the pre-heating time necessary for reactivating CK activity and the study on the CK activating ability in the stored serum are performed.
No study has been made on the storage stability of the reagent for measuring CK activity using the compound as a CK activator in a solution state. However, as described in Japanese Patent Publication No. 5-38600 (column 3, line 39 to column 4, line 18),
These reagents for measuring CK activity have poor storage stability in a solution state, and the room temperature (18 to 37)
C)), and it was considered by those skilled in the art as having a very short life and having to be used in a short time. Also,
In each country, recommended methods for measuring CK activity are selected by various organizations such as various academic societies, and the type of CK activator and the like are selected along with various measurement conditions. For example, reduced glutathione (hereinafter abbreviated as GSH) was selected by the German Society of Clinical Chemistry (GSCC) in 1970, and 197 was further selected.
In the seven years, among 27 types of SH compounds including the conventional SH compounds as described above, activating ability, stability in a dry state and after dissolution, presence or absence of interference by other serum enzymes, coexistence with serum, Considering the presence / absence of turbidity, odor and price, NAC has been adopted as a CK activator [Clin. C
hem. Clin. Biochem., Vol. 15, 255-260 (1977)]. However, since NAC is relatively unstable, the SH group of NAC is gradually oxidized in a solution. Therefore, NA
When C is used as a CK activator in a reagent for measuring CK activity, the effect as a CK activator cannot be maintained for a long time. Furthermore, since the oxidation product of NAC has the property of inhibiting CK activity, [Scand. J. Clin. Lab. Invest.
39, 737-742 (1979)], and these properties of NAC itself are a major cause of inhibiting the storage stability of the reagent for measuring CK activity in a solution state. After that, various studies have been made to address the above-mentioned problems when NAC is used as a CK activator. Ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) is used as a reagent for measuring CK activity.
It was found that the addition of NAC increases the stability of NAC and suppresses the formation of oxides having a CK activity inhibitory action. Based on these findings, the Scandinavian Society of Clinical Chemistry and the Society of Clinical Physiology (SSCC & CP) and the Japanese Society of Clinical Chemistry (JSCC) have adopted a combination of EDTA and NAC, as well as the International Clinical Chemistry Union (IFCC). The combination of EDTA and NAC was adopted [Clinical Chemistry,
19, No. 2, 184-208 (1990), and Ann. Bio
l. Clin., 48, P185-202 (1990)]. However, the stability of a reagent for measuring CK activity using EDTA and NAC in combination in a solution state is not necessarily sufficient. Therefore, a method of coexisting NAC and an SH compound other than NAC has also been proposed (Japanese Patent Publication No. 5-38600). However, this method cannot sufficiently improve the stability of the reagent for measuring CK activity. could not.

On the other hand, recent trends in clinical diagnostic agents are caused by problems in conventional reagents for clinical tests supplied in a lyophilized state, that is, due to the labor required for preparing reagents and the complexity of preparing reagents. Lyophilized reagents are appropriately dissolved in a buffer or the like for the purpose of improving work efficiency, cost increase, etc. due to reagent preparation mistakes, and to enable quick response at the time of emergency testing. Liquid reagents that do not require the labor of preparing reagents and can be stored for a long period of time have become the mainstream of current clinical diagnostic agents instead of conventional reagents that are prepared and prepared. At present, there is a demand for the development of liquid reagents that can be stored.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and has been made in consideration of the above circumstances, and is intended for CK activation for a liquid reagent which is stable for a long period of time and does not generate an oxidation product having a CK activity inhibiting action. It is an object of the present invention to provide a liquid reagent for measuring CK activity which can be stored for a long period of time using the agent and the activator.

[0006]

Means for Solving the Problems (1) The present invention relates to TG,
2-mercaptoethanol (hereinafter abbreviated as 2ME) and 2-mercaptoethanesulfonic acid (hereinafter, referred to as 2ME)
Abbreviated as 2MES. Or CK for a liquid reagent comprising at least one compound selected from the group consisting of
It is an invention of an activator. (2) Further, the present invention provides a CK activator for a liquid reagent comprising at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof (provided that ethylene glycol or propylene glycol is Except for the case of coexistence). (3) The present invention further provides a CK activity for a liquid reagent which is stable at 10 ° C. for at least 3 months, comprising at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof. It is an invention of an agent. (4) Furthermore, the present invention provides TG, 2ME, and 2ME
It is an invention of a CK activator for a liquid reagent, characterized by containing an SH compound consisting of at least one compound selected from the group consisting of S and salts thereof.

(5) The present invention also relates to a method for measuring CK activity, characterized by containing an SH compound consisting of at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof. Liquid reagent (excluding the case where ethylene glycol or propylene glycol coexists). (6) The present invention further comprises an SH compound consisting of at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof, at least at 10 ° C. CK that is stable for 3 months
It is an invention of a liquid reagent for activity measurement.

(7) The present invention further relates to adenosine diphosphate (hereinafter abbreviated as ADP), hexokinase (hereinafter abbreviated as HK), or glucokinase (hereinafter abbreviated as GK), Nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter, NAD)
Abbreviated as ADP. ), Glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH), and an SH compound comprising only one or more compounds selected from the group consisting of TG, 2ME, and 2MES or a salt thereof. PH of 7.5 to 1 containing one reagent and creatine phosphate (hereinafter abbreviated as CP).
The present invention is directed to a liquid reagent for measuring creatine kinase activity, comprising: a first reagent and a second reagent, each of which comprises glucose and magnesium ions.

(8) The present invention also provides an imidazole-acetate buffer, magnesium acetate, sodium azide, NA
DP, HK, G6PDH, ADP, adenosine monophosphate (hereinafter abbreviated as AMP.), TG, diadenosine phosphorus pentachloride acid (hereinafter abbreviated as AP ₅ A.), Glucose,
And EDTA, and a first reagent comprising N, N-bis (2-hydroxyethyl) glycine (hereinafter referred to as Bicin
Abbreviated as e. )-CK activity measurement, characterized by combining a sodium hydroxide buffer, magnesium acetate, sodium azide, EDTA, glucose and CP with a second reagent having a pH of 7.5 to 10. Liquid reagent kit (excluding the case where an SH compound other than thioglycerol coexists).

That is, the present inventors have proposed that C
As a result of intensive studies for an SH compound that can be used as a K activator, among the SH compounds conventionally known as a CK activator, TG, 2ME and / or 2ME
If S (or a salt thereof) is used as a CK activator, a liquid reagent for measuring CK activity that is unexpectedly stable for a long period of time and does not cause CK activity inhibition by oxidation products of the CK activator can be obtained. And completed the present invention.

The CK activator of the present invention is used by adding it as a CK activator to a liquid reagent used in a per se known CK activity measuring method for reactivating CK activity and measuring CK activity. Is possible. The CK activity measurement method which can use the liquid reagent for CK activity measurement using the CK activator of the present invention includes the following reaction catalyzed by CK.

[0012]

(Equation 1) Or AD generated by a rightward reaction (forward reaction)
A method for quantifying P, for example, a method for measuring CP based on an increase in inorganic phosphoric acid generated by hydrolysis of CP,
DP is pyruvate kinase (hereinafter abbreviated as PK)
Is reacted with phosphoenolpyruvate (hereinafter abbreviated as PEP), and the resulting pyruvate is further reacted with NADH in the presence of lactate dehydrogenase to give NAD.
Ultraviolet method to measure as decrease in H (Tanzer-Gi
lvarg method), a method in which ADP is reacted with PEP in the presence of PK, and pyruvic acid generated is further reacted with 2,4-dinitrophenylhydrazine, and the resulting hydrazone is colorimetrically quantified (Nuttal-Wedin method), etc. Or a method for quantifying creatine or ATP produced by a leftward reaction (reverse reaction) of the above reaction catalyzed by CK, for example, a method for colorimetric quantification of creatine using a β-naphthol-diacetyl reaction (Hughes
Method), a method of reacting creatine with ninhydrin and measuring the fluorescence of a condensate of creatine and ninhydrin (Conn-Anido method), a method using luciferase (JP-A-51-41597, JP-A-55-41597). 120796, JP-A-56-26200, JP-A-57-105199), a method using phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (JP-B-59-34119, JP-A-56-155000.),
HK, a method using G6PDH, that is, ATP is converted to HK
Reacting with glucose in the presence of
6-phosphate (G6P) and NA in the presence of G6PDH
NADH produced by reaction with D (or NADP)
(Or NADPH) at 340 nm. Among these known measuring methods, the above-mentioned measuring method is excellent in principle, has good sensitivity and reproducibility, and is a method capable of processing a large number of samples with an automatic analyzer. It is particularly preferable as a method for measuring CK activity using a liquid reagent for measuring CK activity using an agent.

The liquid reagent for measuring CK activity according to the present invention comprises CK
Except for using at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof as an activator, a reagent generally used in a known CK activity measurement method as described above is used. Is enough.

In the present invention, any of the above compounds used as CK activators, even when added in high concentration,
No K activity inhibition is observed. Therefore, the above compound is
When used as a liquid reagent for K activity measurement, the concentration used in the reaction solution at the time of CK activity measurement is not particularly limited as long as it is higher than the concentration at which CK activating ability is generated. For example, TG
Is used as the CK activator of the liquid reagent for measuring CK activity, the concentration used in the liquid reagent for measuring CK activity is such that the concentration in the reaction solution at the time of measuring CK activity is usually 10 mM or more. However, considering economics, it is preferably appropriately selected from the range of 10 to 260 mM. Also,
When 2ME is used as the CK activator, the concentration used in the reaction solution at the time of measuring the CK activity is usually 5 mM.
It may be added to the liquid reagent for measuring CK activity as described above, but in consideration of economy, preferably 5 to 80 mM
Is appropriately selected from the range. CK with 2MES or its salt
When used as an activator, the concentration used is CK
What is necessary is just to add to the CK activity measurement liquid reagent so that the density | concentration in the reaction liquid at the time of an activity measurement may become 5 mM or more normally.
In consideration of economy, it is preferably appropriately selected from the range of 5 to 80 mM. In addition, as a salt of 2MES, for example, Na
Preferred are alkali metal salts such as salts, K salts, and Li salts. In using the CK activator of the present invention, it goes without saying that the above compounds may be used alone or in combination as appropriate.

The liquid reagent for measuring CK activity according to the present invention includes at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof as a CK activator, for example, CP, ADP, glucose, HK or G
Representative examples include those prepared using K, NAD (or NADP), G6PDH, magnesium ion, and the like as main components. The CK of the present invention
In the liquid reagent for activity measurement, for example, EDTA, diaminocyclohexanetetraacetic acid monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA-OH), ethylenediamine It is preferable to contain a chelating agent such as acetic acid (EDDA) and hydroxyethyliminodiacetic acid (HIDA) or a salt thereof (alkali metal salt, ammonium salt, etc.). If necessary, a preservative such as sodium azide, for example, polyoxyethylene octyl phenyl ether [for example, Triton X-100: manufactured by Rohm and Haas Co.], polyoxyethylene lauryl ether [for example, Emulgen 120: Manufactured by Kao Corporation] and the like, for example, imidazole buffer, bis (2-hydroxyethyl) iminotris-
Buffers such as (hydroxymethyl) methane (Bis-Tris) buffer, for example, AMP, diadenosine tetraphosphate (hereinafter A
Abbreviated as P ₄ A. ), AP ₅ A, diadenosine hexaphosphate (hereinafter, abbreviated as AP ₆ A.) Of di-adenosine polyphosphate such as adenylate kinase to give a positive error to the CK activity present body fluid sample (hereinafter, Abbreviated as AK.)
It goes without saying that reagents usually used in the method for measuring CK activity, such as AK inhibitors for avoiding the influence of the activity, may be contained in the concentration range usually used in this field.

The origin of the HK according to the present invention is not particularly limited, but, for example, those derived from microorganisms and those derived from animals can be used. GK having a higher substrate specificity for glucose than HK can also be used in the same manner as HK, and its origin is not particularly limited. For example, those derived from microorganisms such as Bacillus, those derived from animals Things can be used. These HK and GK are C
It may be added to the CK activity measurement liquid reagent so that the concentration in the reaction solution at the time of the K activity measurement falls within the concentration range used in the measurement method known per se. The medium concentration is usually 0.5 u / ml to 20 u / ml, preferably 0.5 u / ml.
It is added to the liquid reagent for measuring CK activity so as to be u / ml to 5.0 u / ml. When the ammonium sulfate concentration becomes about 50 mM, CK
It is known that the activity is reduced by 10 to 15%.
HK or GK added to the liquid reagent for K activity measurement is
It is not preferable to use the ammonium sulfate suspension as it is.

Since the CK activity inhibition is recognized when the CP according to the present invention is used in the presence of a high concentration, the concentration in the reaction solution at the time of measuring the CK activity is usually 5 mM to 70 mM, preferably 30 mM to 50 mM.
It is added to the liquid reagent for measuring CK activity so as to be mM. Incidentally, since CP is known to be stable in an alkaline solution, the pH of the test solution containing CP is usually pH 7.5 to 1
The pH is appropriately adjusted so as to be in the range of 0, preferably pH 8 to 9.5.

The source of G6PDH according to the present invention is not particularly limited.
Those derived from microorganisms such as mesenteroides (Leuconostoc mesenteroides) and baker's yeast, and those derived from animals can be used. G6PDH may be added to the liquid reagent for CK activity measurement so that the concentration in the reaction solution at the time of measuring CK activity falls within the concentration range used in a measurement method known per se. Usually 0 as concentration.
It is added to the liquid reagent for measuring CK activity so as to have a concentration of 5 u / ml to 40 u / ml, preferably 1 u / ml to 10 u / ml. It is known that when the ammonium sulfate concentration becomes about 50 mM, the CK activity is reduced by 10 to 15%. Therefore, as the G6PDH to be added to the liquid reagent for measuring the CK activity, the G6PDH in a suspended state of ammonium sulfate is used as it is. It is less preferred to do so. In addition, glucose dehydrogenase or NA is contained in G6PDH to be used.
It should be noted that the presence of DH (or NADPH) oxidase may cause deterioration of the reagent or increase of the blind value.

The NAD (or NADP) and ADP according to the present invention are added to the liquid reagent for measuring CK activity such that the concentration in the reaction solution at the time of measuring CK activity falls within the concentration range used in a method known per se. NAD (or NAD
P) is usually 0.1% as the concentration in the reaction solution when measuring CK activity.
The ADP is added to the CK activity measurement liquid reagent so as to have a concentration of 1 mM to 10 mM, preferably 1 mM to 5 mM.
Usually 0.1 mM to 10 mM as the concentration in the reaction solution at the time of activity measurement,
Preferably, it is added to the liquid reagent for measuring CK activity so as to have a concentration of 1 mM to 5 mM.

Glucose used as a substrate for HK or GK according to the present invention is used in the liquid reagent for measuring CK activity so that the concentration in the reaction solution at the time of measuring CK activity is usually 1 mM to 200 mM, preferably 5 mM to 100 mM. Is added to

The magnesium ion according to the present invention is not particularly limited. For example, all magnesium ions derived from magnesium salts usually used in this field, such as magnesium acetate, magnesium chloride and magnesium sulfate, can be used. Since CK activity is known to be non-competitively inhibited by anions, it is desirable to use magnesium acetate, magnesium chloride, or the like that dissociates anions having a small degree of inhibition as a source of magnesium ions. The use concentration of these magnesium salts cannot be unconditionally determined because they slightly differ depending on the kind, but is appropriately selected from the range of usually 2 to 30 mM, preferably 5 to 25 mM. Specifically, for example, when magnesium acetate is used, it is considered that a high concentration may cause a decrease in CK activity, and may affect the measured value of CK activity in correlation with ADP or buffer concentration. Then, CK
It is added to the liquid reagent for measuring CK activity so that the concentration in the reaction solution at the time of activity measurement is usually 2 mM to 30 mM, preferably 8 mM to 20 mM. In addition, it goes without saying that the above-mentioned magnesium salts, which are the supply sources of magnesium ions of the present invention, may be used alone or in an appropriate combination.

The liquid reagent for measuring CK activity according to the present invention comprises ADP, HK (or GK), NAD (or NAD).
P), G6PDH, TG, 2ME and 2MES or a first reagent consisting of at least one compound selected from the group consisting of salts thereof, and a pH consisting of CP etc. of 7.5-1.
A combination of the first and second reagents, wherein each of glucose and magnesium ions is contained in at least one of the first and second reagents. . The following combinations may be mentioned as to which of the first and second reagents contains glucose and magnesium ions.

[0023] In the measurement of CK activity in a two-reagent system, when various coexisting substances (for example, bilirubin, hemoglobin, ascorbic acid, lactic acid, AK, etc.) in a body fluid serving as a sample affect the measurement system. CK with higher accuracy
In order to perform the activity measurement, a method of subtracting the sample blinding, measurement by the so-called double kinetic method is more preferable,
When measurement is performed by this method, glucose and magnesium ions must be contained at least in the first reagent (that is, a combination of 3, 4, 8, and 9 in the above table).
It is known that CP is stable in an alkaline solution.
pH on the alkaline side, for example, usually 7.5 to 10, preferably 8 to
It is desirable to set it to 9.5. However, when the first reagent and the second reagent are mixed, that is, the pH at the time of measuring the CK activity is the optimum pH of CK.
H, for example, it is necessary to appropriately adjust the buffer and the buffer concentration of the first reagent and the second reagent so as to be in the range of pH 6.0 to 7.2, and further, it inhibits CK activity when measuring CK activity. It goes without saying that buffers and buffer concentrations are not preferred. As a buffer which can be used for a reagent containing CP for such a purpose, for example, Bicine, N- [tris (hydroxymethyl) methyl] glycine and the like are preferably mentioned. The concentration of the buffer used in the present invention cannot be specified unconditionally because it differs depending on the type of the buffer used, but the concentration of the buffer used in the first test solution is usually 40 mM to 250 mM. , Preferably in the range of 80 mM to 150 mM, and the concentration of the buffer used in the second reagent is usually 1 mM to 150 mM, preferably 1 mM to 30 mM.
Each is appropriately selected so as to be within the range of M. The pH of the first reagent is usually 6.0 to 7.2, preferably 6.4 to 7.0.
And the pH of the second reagent is appropriately selected so as to be usually in the range of 7.5 to 10, preferably 8 to 9.5. For example, when imidazole is used as a buffer of the first reagent, as the concentration in the first reagent,
Usually, the concentration is in the range of 60 mM to 250 mM, preferably 80 mM to 150 mM, and when Bicine is used as a buffer for the second reagent, the concentration in the second reagent is usually 1 mM to
Each may be appropriately selected so as to be in the range of 100 mM, preferably 1 mM to 30 mM.

In order to measure the CK activity using the liquid reagent for measuring the CK activity of the present invention, it is sufficient to carry out the measurement according to a method known per se.

The CK activator of the present invention can be used for various other SHs.
Compounds such as NAC, DTT, glutathione, L-
Although conventionally known as a CK activator together with cysteine and the like, among various SH compounds known as the CK activator, the oxidation product thereof is CK.
Surprisingly, only TG, 2ME, and 2MES or a salt thereof according to the present invention did not inhibit the activity and could maintain a stable CK activating ability in an aqueous solution for a long period of time. The present inventors have prepared a liquid reagent for measuring CK activity of the present invention containing these TG, 2ME, and 2MES or a salt thereof based on such findings first discovered by the present inventors. Long-term stability in a solution state, which has been considered difficult with the reagent for measuring CK activity of
When stored at 10 ° C., they found that they could be used without deteriorating the performance for at least three months, usually six months or more, and completed the present invention.

The liquid reagent kit for measuring CK activity of the present invention is a liquid reagent kit used for measuring CK activity in a body fluid such as serum, which can be stored for a long period of time. Magnesium, sodium azide, NADP, HK, G6PDH, ADP, AMP,
Including TG, a first reagent AP ₅ A, comprising glucose, and EDTA, a pH6.0~7.2, Bicine- sodium hydroxide buffer, magnesium acetate, sodium azide, EDTA, glucose, and CP, the PH
It is a combination of 7.5 to 10.0 second reagents, and preferred embodiments, specific examples, and the like of each component are as described above. Needless to say, the kit may be combined with a standard CK product or the like as necessary.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples, and Comparative Examples, but the present invention is not limited thereto.

[0028]

【Example】

Reference Example 1. Examination of CK activity inhibitory effect of oxidation product of SH compound [oxidation product] TG, 2ME, 2MES (sodium salt), NAC, GSH, mercaptosuccinic acid, 1-thio-β-D-glucose disodium Salt dihydrate (TD
G) each of 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate (AHMP), 2-amino-6-mercaptopurine riboside hydrate (AMPR), thiophenol, 2-thiouracil and DTT SH compound
The 300 mM aqueous solution was left at a high temperature until no SH group was detected to obtain an oxidation product of each SH compound. [Enzyme solution] 128 mM imidazole-acetate buffer (pH 6.7)
In addition, 26 mM of NAC as a CK activator, 5 mM of each oxidation product of each SH compound obtained above, 2.6 mM of ADP,
K (manufactured by Oriental Yeast) 3.9 u / ml, G6PDH (manufactured by Oriental Yeast) 8.0 u / ml, NADP 2.6 mM, glucose 21 mM, magnesium acetate 10 mM, AMP 6.5 mM,
AP ₅ A 0.013 mM, sodium azide 0.1% (W / V), E
After dissolving to 2 mM DTA-2Na, the pH was adjusted to 6.7 with sodium hydroxide, and this was used as an enzyme solution.
Also, an enzyme solution containing no SH compound oxidation product was prepared,
This served as a control. [Substrate solution] 10 mM Bicine-sodium hydroxide buffer (pH
8.5), CP 155 mM, glucose 21 mM, magnesium acetate 10 mM, sodium azide 0.1% (W / V), EDTA
After dissolving to a concentration of -2Na 2 mM, the solution was adjusted to pH 9.0 with sodium hydroxide to obtain a substrate solution. [Sample] Five samples of fresh human serum were used. [Measurement of CK activity] 320 µl of the enzyme solution was added to 8 µl of the sample, preliminarily heated for 5 minutes, and then 80 µl of the substrate solution was further added to start the reaction. The reaction was started for 2 minutes after the addition of the substrate solution for 3 minutes. The change in absorbance per minute was determined, and the CK activity value was calculated from the molecular extinction coefficient of the generated NADPH. The measurement was performed using a Hitachi 7150-type automatic analyzer, measuring at a main wavelength of 340 nm and a sub-wavelength of 405 nm at a measurement temperature of 37 ° C. Tables 1 and 2 show the CK activity values of the five types of specimens and their average values, respectively.

[0029]

[Table 1]

[0030]

[Table 2] *% Indicates a relative value when the average value of CK activity measured using an enzyme solution without the SH compound oxidation product immediately after preparation is 100%.

As is clear from the results in Tables 1 and 2, among the SH compounds, the CK activator T
G, 2ME and 2MES oxidation products, and TDG;
It can be seen that the oxidation products of AHMP, 2-thiouracil and DTT hardly inhibit CK activity.

Example 1. [CK activator] TG, 2ME and 2MES, and T
G + 2ME was used as the CK activator. [Enzyme solution] 128 mM imidazole-acetate buffer (pH 6.7)
In addition, an SH compound appropriately selected from the above-mentioned CK activator, ADP 2.6 mM, HK (manufactured by Oriental Yeast) 3.9 u / ml, G6PDH (manufactured by Oriental Yeast) 8.0
u / ml, NADP 2.6mM, glucose 21 mM, magnesium acetate _{10mM, AMP 6.5mM, AP 5 A} 0.013mM, sodium azide 0.1% (W / V), was dissolved at a EDTA-2Na 2 mM, hydroxide A solution adjusted to pH 6.7 with sodium and stored at a predetermined temperature for a predetermined period was used as an enzyme solution. [Substrate solution] 10 mM Bicine-sodium hydroxide buffer (pH
8.5), CP 155 mM, glucose 21 mM, magnesium acetate 10 mM, sodium azide 0.1% (W / V), EDTA
After dissolving to a concentration of -2Na 2 mM, the solution was adjusted to pH 9.0 with sodium hydroxide to obtain a substrate solution. [Sample] Five samples of fresh human serum were used. [Measurement of CK activity] 320 µl of the enzyme solution was added to 8 µl of the sample, preliminarily heated for 5 minutes, and then 80 µl of the substrate solution was further added to start the reaction. The reaction was started for 2 minutes after the addition of the substrate solution for 3 minutes. The change in absorbance per minute was determined, and the CK activity value was calculated from the molecular extinction coefficient of the generated NADPH. The measurement was performed using a Hitachi 7150-type automatic analyzer, measuring at a main wavelength of 340 nm and a sub-wavelength of 405 nm at a measurement temperature of 37 ° C. Table 3 shows the CK activity values of the five types of specimens and their average values.

Comparative Example 1. [CK Activator] From the results of Reference Example 1, TDG and D are SH compounds whose oxidation products do not inhibit CK activity.
TT, AHMP, 2-thiouracil, and the conventional CK activators NAC, L-cysteine, GSH, and NAC + GSH, NAC + TG and NAC + 2ME
K activator. [Enzyme solution] Performed using the same reagent as in Example 1 except that a predetermined concentration of an SH compound appropriately selected from the above CK activating agents was used instead of the CK activating agent used in Example 1. Prepared as in Example 1. [Substrate solution] Same as in Example 1. [Specimen] Same as in Example 1. [Measurement of CK activity] It was measured in the same manner as in Example 1. Tables 3 and 4 show the CK activity values of the five types of specimens and their average values, respectively. Tables 3 and 4 do not describe the results obtained when AHMP and 2-thiouracil were used as CK activators, but these compounds had poor solubility and sufficient CK activity was obtained. Since there was no data, the data was omitted.

[0034]

[Table 3]

[0035]

[Table 4] *% Indicates NAC as the CK activator in Comparative Example 1.
Shows the relative value when the average value of CK activity measured using the enzyme solution immediately after preparation with the addition of 100% is taken as 100%.

As is clear from the results shown in Tables 3 and 4, the CK activator according to the present invention has a conventionally used NA.
It turns out that it has CK activation ability equivalent to C. Further, when added to an aqueous solution, the CK activating agent according to the present invention, which can maintain the CK activating ability for a long period of time, can be used among various SH compounds including NAC.
E and 2MES are also found. Further, from the results in Table 4, when NAC and TG were used in combination as CK activators, CK after storage at 10 ° C. and 20 ° C. for one month was used.
Activating ability is reduced, and when NAC and 2ME are used in combination, C is maintained even after storage at 10 ° C. for one month.
It can be seen that the K activating ability is stable, but the CK activating ability after storage at 20 ° C. for one month is clearly reduced. From these facts, it is understood that it is difficult to prepare a long-term stable liquid reagent for CK activity measurement by the conventional concept on the assumption that NAC is used as the main component of the CK activator. Considering the results of Reference Example 1, even if the oxidation product does not inhibit CK activity,
Inappropriate CK activators for liquid reagents for measuring K activity, for example, those which cannot maintain the CK activating ability in a solution such as TDG or DTT for a long period of time, for example, AHMP It can be seen that there are some compounds such as thiouracil and 2-thiouracil which do not show sufficient CK activating ability as a result of solubility problems and the like. From these facts, as a CK activator for a liquid reagent requiring storage stability for a long period of time, a CK activator having at least sufficient CK activating ability, a CK activator which is stable in an aqueous solution for a long period of time, It is considered necessary to be able to retain the activating ability and further to have properties such that the oxidation product does not inhibit the CK activity, but TG, 2ME and 2MES according to the present invention are: It turns out that all these conditions are satisfied.

Example 2 [Enzyme solution] The enzyme solution prepared in Example 1 and containing 100 mM TG as a CK activator was used. [Substrate solution] Same as in Example 1. [Sample] For each measurement, 10 samples of different fresh human serum were used. [Measurement of CK activity] The enzyme solution and the substrate solution were stored at 10 ° C for a predetermined period, and the measurement was carried out in the same manner as in Example 1 except that the above-mentioned sample was used. Ten
Table 5 shows the average value of each CK activity value of each type of specimen.

Comparative Example 2 [Enzyme solution] The same reagent as in Example 1 was used in the same manner as in Example 1 except that 26 mM NAC was used instead of the CK activator used in Example 1. The one prepared in this manner was used. [Substrate solution] Same as in Example 1. [Specimen] Same as Example 2. [Measurement of CK activity] The enzyme solution and the substrate solution were stored at 10 ° C for a predetermined period, and the measurement was carried out in the same manner as in Example 1 except that the above-mentioned sample was used. Ten
Table 5 shows the average value of each CK activity value of each type of specimen.

[0039]

[Table 5] * () Indicates the relative value when the average CK activity measured using an enzyme solution (100 mM TG or 26 mM NAC as a CK activator) and substrate solution prepared at the time of use was taken as 100%. Show.

As is evident from the results in Table 5, the conventional CK activity measuring reagent using NAC as the CK activator shows a marked decrease in the measured CK activity after storage at 10 ° C. for 3 months. It turns out that it is difficult to use as a reagent for measuring CK activity. On the other hand, the liquid reagent for measuring CK activity of the present invention showed no effect on the measured value of CK activity even after storage at 10 ° C. for 7 months, indicating that the liquid reagent maintained the performance immediately after preparation. . Further, as a result of studying the calibration range, the liquid reagent for measuring CK activity of the present invention has a calibration range equivalent to that immediately after preparation even after storage at 10 ° C. for 7 months, and shows the performance immediately after preparation. We confirmed that we held.

Example 3 Representative examples of liquid reagent kits that can be stored for a long period of time and are used to measure CK activity in body fluids such as serum include the following. (1) First reagent (pH 6.0 to pH 7.2): imidazole-acetic acid buffer, magnesium acetate, sodium azide, NAD
P, HK, G6PDH, ADP, AMP, TG, AP ₅
A, glucose, EDTA. (2) Second reagent (pH 7.5 to pH 10.0): Bicine-sodium hydroxide buffer, magnesium acetate, sodium azide, EDTA, glucose, CP.

[0042]

Industrial Applicability The present invention provides a CK activator for a liquid reagent and a liquid reagent for measuring CK activity in a body fluid such as serum using the activator. The CK activator of the present invention is stable for a long time and CK
It does not produce an oxidation product having an activity-inhibiting action and can be stored for a long period of time when used as a CK activator for liquid reagents, and at least 3 when stored at a low temperature, for example, at 10 ° C. It has a remarkable effect in that it can be used without deteriorating the performance for more than six months, usually six months.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/50 C12N 9/12 BIOSIS (DIALOG) MEDLINE (STN)

Claims (7)

(57) [Claims] 1. An SH compound comprising at least one compound selected from the group consisting of thioglycerol, 2-mercaptoethanol, and 2-mercaptoethanesulfonic acid or a salt thereof. Liquid reagent for measuring creatine kinase activity (except when ethylene glycol or propylene glycol coexists). 2. Adenosine diphosphate, hexokinase or glucokinase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate dehydrogenase and thioglycerol, 2-mercaptoethanol, and 2-mercaptoethane A first reagent comprising an SH compound consisting solely of at least one compound selected from the group consisting of sulfonic acids or salts thereof, a second reagent comprising creatine phosphate having a pH of 7.5 to 10, and A liquid reagent for measuring creatine kinase activity, wherein each of glucose and magnesium ions is contained in at least one of the first reagent and the second reagent. 3. The liquid reagent for measuring creatine kinase activity according to claim 2 , wherein glucose and magnesium ions are contained in the first reagent. 4. A creatine kinase activity measured for liquid reagent according to claim 2 or 3 magnesium ions are those derived from magnesium acetate and / or magnesium chloride. 5. The first and second reagents is at least three months storage stability at 10 ° C., creatine kinase activity measurement for liquid reagent according to any one of claims 2-4. 6. An imidazole-acetate buffer, magnesium acetate, sodium azide, nicotinamide adenine dinucleotide phosphate, hexokinase, glucose-6
A first reagent comprising phosphate dehydrogenase, adenosine diphosphate, adenosine monophosphate, thioglycerol, diadenosine pentaphosphate, glucose and ethylenediaminetetraacetic acid, and N, N-bis (2-hydroxyethyl ) Glycine-sodium hydroxide buffer, magnesium acetate, sodium azide, ethylenediaminetetraacetic acid,
A liquid reagent kit for measuring creatine kinase activity (provided that an SH compound other than thioglycerol is used), which is characterized by being combined with a second reagent having a pH of 7.5 to 10 containing glucose and creatine phosphate. Except when they coexist.). 7. The liquid reagent kit for measuring creatine kinase activity according to claim 6 , wherein the first reagent and the second reagent are storage-stable at 10 ° C. for at least 3 months.