JPH08242893A - Liquid reagent for determination of creatine kinase activity - Google Patents

Abstract

PURPOSE: To provide the subject liquid reagent containing one or more compounds selected from thioglycerol, HSC2 H4 OH, HSC2 H4 SO3 H, etc., stably storable over a long period, free from generation of oxidized product having a creatine kinase inhibiting action and useful for the diagnosis of muscular diseases, heart diseases, etc. CONSTITUTION: This liquid reagent for the determination of creatine kinase activity, stably storable over a long period, free from generation of oxidized product having a creatine kinase inhibiting action and useful in a clinical examination field for the diagnosis of muscular diseases, heart diseases, etc., is composed of the 1st reagent containing at least one kind of compound such as thioglycerol, 2-mercaptoethanol, 2-mercaptoethanesulfonic acid (salt), etc., adenosine diphosphate, hexokinase or glucokinase, NAD or NADP and glucose-6- phosphate dehydrogenase and the 2nd reagent containing creatine phosphate and having pH of 7.5-10. Either one or both of the 1st reagent and the 2nd reagent are incorporated with glucose and magnesium ion.

Description

Detailed Description of the Invention

[0001]

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

[0002]

In the living body, CK is most abundant in skeletal muscle, followed by brain and myocardium, and also in smooth muscle and nervous system. not exist. In 1959, patients with progressive muscular dystrophy showed an increase in serum and then became clinically applied. Currently, in the field of clinical examination, measurement of CK activity is performed for muscle diseases,
It is one of the important items that are routinely performed for the diagnosis of heart disease and the like.

On the other hand, since CK is rapidly inactivated in serum, when measuring CK activity, an activator was added to an activity measuring reagent and the CK was activated by the activator. It is common to make measurements later. As an activator of CK, so-called SH group-containing compounds (hereinafter abbreviated as SH compounds) such as N-acetyl cysteine (hereinafter abbreviated as NAC), dithiothreitol (hereinafter abbreviated as DTT), glutathione, and the like. However, as the CK activator, various SHs have been used so far.
Compounds have been investigated. For example, 1968 Hess
Is cysteine, 1972 Warren is DTT,
1976 Szats used NAC as CK activators, respectively [Clinical Chemistry, Vol. 19, No. 2, 184].
~ 208 (1990), and Clin. Chem., Vol. 23, 1569 ~ 1575
(1977)]. Furthermore, Morin in 1977 announced that thioglycerol (hereinafter abbreviated as TG) and mercaptoethanol are suitable as CK activators [Clin. Chem., Vol. 23, 1569-1575 (1977)]. . However, in this document, only the preliminary heating time required for reactivating CK activity and the ability to activate CK in stored serum have been examined, and these SH
The storage stability of a reagent for measuring CK activity using a compound as a CK activator in a solution state has not been studied at all. However, as described in Japanese Examined 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 reagent at room temperature (18 to 37
It was considered by those skilled in the art that it has a very short life at (° C.) and must be used in a short time. Also,
In various countries, various organizations such as various academic societies have selected the recommended method for measuring CK activity, and the kinds of CK activators are selected along with various measurement conditions. For example, reduced glutathione (hereinafter abbreviated as GSH) was selected by the German Society for Clinical Chemistry (GSCC) in 1970, and 197
In 7 years, from 27 kinds of SH compounds including the above-mentioned conventional SH compounds, activation ability, stability in dry state and after dissolution, presence or absence of interference by other serum enzymes, coexistence with serum As a result of considering turbidity, odor, price, etc., NAC has been adopted as a CK activator [Clin.C.
hem.Clin.Biochem., Vol.15, 255-260 (1977)]. However, since NAC is relatively unstable, SH groups of NAC are gradually oxidized in the solution. Therefore, NA
When C is used as the CK activator in the reagent for measuring CK activity, the effect as the 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.,
Vol.39, 737-742 (1979)], these properties of NAC itself are a major cause of impairing the storage stability of the reagent for measuring CK activity in a solution state. After that, various studies have been conducted to address the above problems when NAC is used as a CK activator, and ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) is used as a reagent for measuring CK activity.
It was found that the addition of N.sub.2 increases the stability of NAC and suppresses the formation of oxides having a CK activity inhibiting action. Based on the results of these studies, the Scandinavian Society of Clinical Chemistry and the Society of Clinical Physiology (SSCC & CP) and the Japanese Society of Clinical Chemistry (JSCC) adopted the combination of EDTA and NAC, and the International Union of Clinical Chemistry (IFCC) as well. Adopted the combination of EDTA and NAC [Clinical Chemistry,
Volume 19, Issue 2, 184-208 (1990), and Ann.Bio
L. Clin., 48, P185-202 (1990)]. However, the stability of the reagent for measuring CK activity, which uses EDTA and NAC in combination, in a solution state is not always sufficient. Therefore, a method of coexisting NAC and an SH compound other than NAC has also been proposed (Japanese Patent Publication No. 5-38600), but this method also does not sufficiently improve the stability of the CK activity measuring reagent. could not.

On the other hand, as a trend of clinical diagnostic agents in recent years, it is caused by problems in conventional clinical test reagents supplied in a freeze-dried state, that is, labor of reagent preparation and complexity of reagent preparation. Dissolve freeze-dried reagents with appropriate buffers, etc. for the purpose of improving work efficiency and cost increase due to mistakes in reagent preparation, and for enabling quick response during emergency tests. Liquid reagents that can be stored for a long period of time without the need for reagent preparation have become the mainstream of current clinical diagnostic agents instead of conventional reagents prepared by At present, there is a demand for the development of a liquid reagent that can be stored.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above situation, and is CK activation for a liquid reagent which is stable for a long period of time and does not produce an oxidation product having a CK activity inhibiting action. An object of the present invention is to provide a liquid reagent for CK activity measurement, which uses the agent and the activator and can be stored for a long period of time.

[0006]

[Means for Solving the Problems]

(1) The present invention is at least selected from the group consisting of TG, 2-mercaptoethanol (hereinafter abbreviated as 2ME), 2-mercaptoethanesulfonic acid (hereinafter abbreviated as 2MES), or a salt thereof. It is an invention of a CK activator for a liquid reagent, which comprises one or more compounds.

(2) Further, the present invention is characterized by containing at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof.
It is an invention of a liquid reagent for measuring K activity.

(3) Further, according to the present invention, adenosine diphosphate (hereinafter abbreviated as ADP), hexokinase (hereinafter abbreviated as HK) or glucokinase (hereinafter abbreviated as GK), nicotine. Amidoadenine dinucleotide (hereinafter abbreviated as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter NAD)
Abbreviated as P. ), Glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH) and T.
G, 2ME, and 2MES or a salt thereof, and a first reagent containing at least one compound selected from the group consisting of, and creatine phosphate (hereinafter abbreviated as CP), and a pH of 7.5 to. In the invention of a liquid reagent for measuring CK activity, which comprises 10 second reagents, wherein each of glucose and magnesium ion is contained in at least one of the first reagent and the second reagent. is there.

(4) Furthermore, the present invention provides an imidazole-acetic acid buffer, magnesium acetate, sodium azide,
It comprises NADP, HK, G6PDH, ADP, adenosine monophosphate (hereinafter abbreviated as AMP), TG, diadenosine pentaphosphate (hereinafter abbreviated as AP ₅ A), glucose, and EDTA. First reagent, N, N-
Bis (2-hydroxyethyl) glycine (hereinafter referred to as Bic
Abbreviated as ine. ) -For measuring CK activity, characterized in that it comprises a combination of a second reagent having a pH of 7.5 to 10 comprising a sodium hydroxide buffer solution, magnesium acetate, sodium azide, EDTA, glucose and CP. It is an invention of a liquid reagent kit.

That is, the present inventors have found that C
As a result of intensive studies for SH compounds usable as K activators, TG, 2ME or / and 2ME among SH compounds conventionally known as CK activators have been obtained.
When S (or its salt) is used as a CK activator, it is possible to obtain a liquid reagent for CK activity measurement which is unexpectedly stable for a long period of time and does not cause CK activity inhibition by an oxidation product of the CK activator. The present invention has been completed and the present invention has been completed.

The CK activator of the present invention should be used by adding it as a CK activator to a liquid reagent used in a known CK activity measuring method for reactivating CK activity to measure CK activity. Is possible. As the CK activity measuring method in which the liquid reagent for CK activity measurement using the CK activator of the present invention can be used, the following reaction catalyzed by CK

[0012]

(Equation 1) CP or AD generated by the rightward reaction (positive reaction) of
A method for quantifying P, for example, a method for measuring CP based on an increase in inorganic phosphate produced by hydrolysis of CP, A
DP is pyruvate kinase (hereinafter abbreviated as PK)
In the presence of phosphoenolpyruvate (hereinafter abbreviated as PEP), the resulting pyruvate is further reacted with NADH in the presence of lactate dehydrogenase to give NAD.
Ultraviolet method (Tanzer-Gi)
lvarg method), a method in which ADP is reacted with PEP in the presence of PK, and the produced pyruvic acid is further reacted with 2,4-dinitrophenylhydrazine to colorimetrically determine the produced hydrazone (Nuttal-Wedin method), etc. Alternatively, a method for quantifying creatine or ATP produced by the leftward reaction (reverse reaction) of the above reaction catalyzed by CK, for example, a method for colorimetrically quantifying creatine using a β-naphthol diacetyl reaction (Hughes
Method), or a method of reacting creatine with ninhydrin to measure fluorescence of a condensate of creatine and ninhydrin (Conn-Anido method), a method using luciferase (JP-A-51-41597 and JP-A-55-415-). No. 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, G6PDH method, ie ATP HK
Glucose produced by reacting with glucose in the presence of
6-phosphate (G6P) and NA in the presence of G6PDH
NADH produced by reacting with D (or NADP)
(Or NADPH) is measured at 340 nm by an ultraviolet method or the like. Among these known measuring methods, the above-mentioned measuring method is theoretically excellent, has good sensitivity and reproducibility, and moreover, can process a large number of samples with an automatic analyzer, and therefore CK activation of the present invention 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 of the present invention is CK
By using a reagent usually used in the CK activity measuring method known per se as described above, except that at least one compound selected from the group consisting of TG, 2ME, and 2MES or a salt thereof is used as an activator. Is enough.

In the present invention, any of the above compounds used as the CK activator is C even when added at a high concentration.
No K activity inhibition is observed. Therefore, if the above compound is C
The concentration used in the reaction solution at the time of measuring CK activity when used as a liquid reagent for K activity measurement is not particularly limited as long as it is at least a concentration at which CK activation ability is generated. For example, TG
When used as a CK activator of a liquid reagent for CK activity measurement, the concentration used in the liquid reagent for CK activity measurement is such that the concentration in the reaction solution during CK activity measurement is usually 10 mM or more. However, it is preferably selected appropriately from the range of 10 to 260 mM in consideration of economic efficiency. Also,
When using 2ME as a CK activator, the concentration used in the reaction solution when measuring CK activity is usually 5 mM.
It may be added to the liquid reagent for CK activity measurement as described above, but considering economic efficiency, it is preferably 5 to 80 mM.
Is appropriately selected from the range. 2 MES or its salt as CK
When used as an activator, the concentration used is CK
It may be added to the liquid reagent for CK activity measurement so that the concentration in the reaction solution at the time of activity measurement is usually 5 mM or more.
Considering economic efficiency, it is preferably selected appropriately from the range of 5 to 80 mM. As the salt of 2MES, for example, Na
Alkali metal salts such as salts, K salts and Li salts are preferred. In using the CK activator of the present invention, it goes without saying that the above compounds may be used alone or in appropriate combination.

The liquid reagent for measuring CK activity of the present invention includes, as a CK activator, at least one compound selected from the group consisting of TG, 2ME, and 2MES or salts thereof, for example, CP, ADP, glucose, HK or G
Representative ones are prepared by using K, NAD (or NADP), G6PDH, magnesium ion and the like as the main components. The CK of the present invention
In the liquid reagent for activity measurement, for the purpose of stabilizing the SH compound and preventing coloration of the reagent, for example, EDTA, diaminocyclohexanetetraacetic acid monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA-OH), ethylenediamine diamine. It is desirable to contain a chelating agent such as acetic acid (EDDA) or hydroxyethyliminodiacetic acid (HIDA) or salts thereof (alkali metal salts, ammonium salts, etc.). In addition, if necessary, a preservative such as sodium azide, for example, polyoxyethylene octyl phenyl ether [for example, Triton X-100: manufactured by Rohm and Haas], polyoxyethylene lauryl ether [for example, Emulgen 120: Kao Corporation] and other nonionic surfactants such as imidazole buffers, bis (2-hydroxyethyl) iminotris-
Buffering agents such as (hydroxymethyl) methane (Bis-Tris) buffering agents such as AMP and diadenosine tetraphosphate (hereinafter referred to as A
Abbreviated as P ₄ A. ), AP ₅ A, diadenosine hexaphosphate (hereinafter abbreviated as AP ₆ A) and the like, such as diadenosine polyphosphate and the like, which give a positive error to CK activity present in a body fluid sample (hereinafter, It is abbreviated as AK.)
It goes without saying that the reagents normally used in the CK activity measuring method 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 or animals can be used. GK, which has a higher substrate specificity for glucose than HK, can also be used in the same manner as HK, and the origin thereof is not particularly limited. For example, those derived from microorganisms such as Bacillus and those derived from animals. Things can be used. These HK and GK are C
It may be added to the liquid reagent for CK activity measurement so that the concentration in the reaction solution at the time of measuring K activity falls within the concentration range used in a measurement method known per se. For example, the reaction solution at the time of measuring CK activity The usual concentration is 0.5u / ml-20u / ml, preferably 0.5u / ml
It is added to the liquid reagent for CK activity measurement so as to be u / ml to 5.0 u / ml. When the ammonium sulfate concentration reaches about 50 mM, CK
Since it is known to reduce the activity by 10 to 15%, C
HK or GK added to the liquid reagent for measuring K activity is
It is not so preferable to use the ammonium sulfate suspension state as it is.

The CP according to the present invention shows inhibition of CK activity when coexisted at a high concentration. Therefore, the concentration in the reaction solution when measuring CK activity is usually 5 mM to 70 mM, preferably 30 mM to 50 mM.
It is added to the liquid reagent for CK activity measurement so that it becomes mM. Since CP is known to be stable in an alkaline solution, the pH of a test solution containing CP is usually pH 7.5 to 1
It is appropriately adjusted to be 0, preferably pH 8 to 9.5.

The origin of the G6PDH according to the present invention is not particularly limited, and examples thereof include leuconostoc.
Those derived from microorganisms such as 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 such that the concentration in the reaction solution during CK activity measurement falls within the concentration range used in a measurement method known per se. Normally 0 as concentration.
5 u / ml to 40 u / ml, preferably 1 u / ml to 10 u / ml is added to the liquid reagent for CK activity measurement. It is known that when the ammonium sulfate concentration reaches about 50 mM, the CK activity is reduced by 10 to 15%. Therefore, the G6PDH added in the liquid reagent for CK activity measurement should be in the ammonium sulfate suspension state as it is. Doing so is not very desirable. In addition, glucose dehydrogenase and NA are included in G6PDH used.
If DH (or NADPH) oxidase is mixed, it may cause deterioration of the reagent or increase of the blinded value, so it is necessary to be careful.

The NAD (or NADP) and ADP according to the present invention are added to a liquid reagent for measuring CK activity so that the concentration in the reaction solution at the time of measuring CK activity falls within the concentration range used in the assay method known per se. NAD (or NAD
P) is usually 0. 0 as the concentration in the reaction solution when measuring CK activity.
1 mM to 10 mM, preferably 1 mM to 5 mM was added to the liquid reagent for measuring CK activity, and ADP was added to CK.
Usually 0.1 mM to 10 mM as the concentration in the reaction solution at the time of activity measurement,
It is preferably added to the liquid reagent for measuring CK activity so as to be 1 mM to 5 mM.

Glucose used as a substrate for HK or GK according to the present invention is contained in a 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. Added to.

The magnesium ion according to the present invention is not particularly limited, but any of those derived from magnesium salts usually used in this field such as magnesium acetate, magnesium chloride, magnesium sulfate, etc. can be used. Since it is known that CK activity undergoes non-competitive inhibition by anions, it is desirable to use magnesium acetate, magnesium chloride or the like, which dissociates anions having a small degree of inhibition, as a source of magnesium ions. The concentration of these magnesium salts to be used cannot be unequivocally stated because it varies slightly depending on the type, but it is usually selected from the range of 2 to 30 mM, preferably 5 to 25 mM. Specifically, for example, in the case of using magnesium acetate, it should be taken into consideration that a high concentration may lead to a decrease in CK activity, and may affect the CK activity measurement value in correlation with ADP and the buffer concentration. Then CK
It is added to the liquid reagent for CK activity measurement 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. Needless to say, the above-mentioned magnesium salts as the magnesium ion supply source of the present invention may be used alone or in appropriate combination.

The liquid reagent for measuring CK activity of the present invention is ADP, HK (or GK), NAD (or NAD).
P), G6PDH, and TG, a second reagent containing at least one compound selected from the group consisting of 2ME and 2MES or salts thereof, and a pH of CP containing 7.5 to 1
0 in combination with the second reagent, and it is preferable to use it in a two-reagent system in which glucose and magnesium ions are contained in at least one of the first reagent and the second reagent. . The following combinations may be mentioned regarding which of the first reagent and the second reagent contains glucose and magnesium ions.

[0023] When measuring CK activity in a two-reagent system, when various coexisting substances (eg, bilirubin, hemoglobin, ascorbic acid, chyle, AK, etc.) in the body fluid to be tested affect the measurement system. There is also a CK with higher accuracy
In order to measure the activity, it is more preferable to subtract the sample blind test, that is, the so-called double kinetic method,
When performing measurement by this method, glucose and magnesium ions must be contained in at least the first reagent (that is, combinations of 3, 4, 8 and 9 in the above table).
Since CP is known to be stable in an alkaline solution, a two-reagent system reagent is a reagent containing CP.
The pH is 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, when the CK activity is measured, the pH is optimum for CK.
H, for example, it is necessary to appropriately adjust the buffering agent and the buffering agent 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 inhibit the CK activity when measuring the CK activity. It goes without saying that the buffering agent and the buffering agent concentration are not preferable. Preferable examples of the buffering agent that can be used for the reagent containing CP for such a purpose include Bicine and N- [tris (hydroxymethyl) methyl] glycine. The use concentration of the buffer used in the present invention, it can not be said unconditionally because it depends on the type of buffer used, but as the use concentration of the buffer in the first reagent solution, usually 40 mM ~ 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 to be in 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 7.5 to 10, preferably 8 to 9.5. For example, when imidazole is used as a buffer for the first reagent, the concentration in the first reagent is
It is usually in the range of 60 mM to 250 mM, preferably 80 mM to 150 mM, and when Bicine is used as the buffer agent of the second reagent, the concentration in the second reagent is usually 1 mM to
It 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 CK activity of the present invention, this may be carried out according to a procedure known per se.

The CK activator of the present invention can be used in various other SH
Compounds such as NAC, DTT, glutathione, L-
Although it is conventionally known as a CK activator together with cysteine, the oxidation product of various SH compounds known as these CK activators is CK.
Surprisingly, TG, 2ME, and 2MES or salts thereof according to the present invention were the only ones that did not inhibit the activity and could retain the stable CK activation ability in an aqueous solution for a long period of time. The present inventors have prepared a liquid reagent for CK activity measurement of the present invention containing these TG, 2ME, and 2MES or a salt thereof, based on such findings found by the present inventors for the first time. Long-term stability in a solution state that has been difficult with the CK activity measurement reagent of
The present invention has been completed by finding that it can be used for at least 3 months, usually 6 months or more without deterioration in performance when stored at 10 ° C, and completed the present invention.

The liquid reagent kit for measuring CK activity of the present invention is a liquid reagent kit which is used for measuring CK activity in body fluids such as serum and which can be stored for a long period of time. Magnesium, sodium azide, NADP, HK, G6PDH, ADP, AMP,
A first reagent having a pH of 6.0 to 7.2 comprising TG, AP ₅ A, glucose and EDTA, and Bicine-sodium hydroxide buffer, magnesium acetate, sodium azide, EDTA, glucose and CP. PH consisting of
It is a combination of 7.5 to 10.0 of the second reagent, and the preferable modes, specific examples and the like of the respective constituent elements are as described above. Further, it goes without saying that the kit may be combined with a CK standard product or the like, if necessary.

The present invention is described in more detail below 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 action of SH compound oxidation product [oxidation product] TG, 2ME, 2MES (sodium salt), NAC, GSH, mercaptosuccinic acid, 1-thio-β-D-glucose disodium Salt dihydrate (TD
G), 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate (AHMP), 2-amino-6-mercaptopurine riboside hydrate (AMPR), thiophenol, 2-thiouracil and DTT. Of SH compounds
The 300 mM aqueous solution was left under high temperature until SH groups were not detected to obtain oxidation products of each SH compound. [Enzyme solution] 128 mM imidazole-acetate buffer (pH 6.7)
In addition, NAC 26 mM as a CK activator, 5 mM equivalent of each SH compound oxidation product obtained above, ADP 2.6 mM, H
K (manufactured by Oriental Yeast Co.) 3.9u / ml, G6PDH (manufactured by Oriental Yeast Co.) 8.0u / ml, NADP 2.6mM, glucose 21mM, magnesium acetate 10mM, AMP 6.5mM,
AP ₅ A 0.013 mM, sodium azide 0.1% (W / V), E
After dissolving so as to have DTA-2Na 2 mM, the pH was adjusted to 6.7 with sodium hydroxide, and this was used as an enzyme solution.
In addition, 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
-2Na was dissolved to a concentration of 2 mM and then adjusted to pH 9.0 with sodium hydroxide to obtain a substrate solution. [Sample] Five fresh human sera 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, and 2 minutes after the addition of the substrate solution, in 3 minutes. The change in absorbance per minute was obtained, and the activity value of CK was calculated from the molecular extinction coefficient of NADPH produced. A Hitachi 7150 type automatic analyzer was used as the measuring device, and the measurement wavelength was 340 nm for the main wavelength and 405 nm for the sub wavelength, and the measurement temperature was 37 ° C. Tables 1 and 2 show the respective CK activity values of the five types of samples and their average values.

[0029]

[Table 1]

[0030]

[Table 2] *% Indicates a relative value when the CK activity average value measured using the SH compound oxidation product-free enzyme solution immediately after preparation is set to 100%.

As is clear from the results shown in Tables 1 and 2, among the SH compounds, T which is the CK activator according to the present invention is used.
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 CK activators was used at a predetermined concentration, ADP 2.6 mM, HK (manufactured by Oriental Yeast Co., Ltd.) 3.9 u / ml, G6PDH (manufactured by Oriental Yeast Co., Ltd.) 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), EDTA-2Na 2 mM, and then hydroxylated The enzyme solution was adjusted to pH 6.7 with sodium and stored at a predetermined temperature for a predetermined period. [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
-2Na was dissolved to a concentration of 2 mM and then adjusted to pH 9.0 with sodium hydroxide to obtain a substrate solution. [Sample] Five fresh human sera 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, and 2 minutes after the addition of the substrate solution, in 3 minutes. The change in absorbance per minute was obtained, and the activity value of CK was calculated from the molecular extinction coefficient of NADPH produced. A Hitachi 7150 type automatic analyzer was used as the measuring device, and the measurement wavelength was 340 nm for the main wavelength and 405 nm for the sub wavelength, and the measurement temperature was 37 ° C. Table 3 shows the respective CK activity values of the five types of samples and their average values.

Comparative Example 1. [CK Activator] From the results of Reference Example 1, TDG and D which are SH compounds whose oxidation products do not inhibit CK activity.
C. TT, AHMP, 2-thiouracil, and conventional CK activators NAC, L-cysteine, GSH, and NAC + GSH, NAC + TG and NAC + 2ME
K activator. [Enzyme Solution] The same reagent as in Example 1 was used, except that an SH compound appropriately selected from the above CK activators was used at a predetermined concentration instead of the CK activator used in Example 1. Prepared as in Example 1. [Substrate solution] Same as in Example 1. [Sample] Same as in Example 1. [Measurement of CK activity] The same measurement as in Example 1 was performed. Tables 3 and 4 show the respective CK activity values of the five types of samples and their average values. Although Table 3 and Table 4 do not show the results when AHMP and 2-thiouracil are used as CK activators, these compounds have poor solubility and sufficient CK activity can be obtained. The data was omitted because it did not exist.

[0034]

[Table 3]

[0035]

[Table 4] *% Is NAC as a CK activator in Comparative Example 1.
The relative value when the average value of CK activity measured using the enzyme solution immediately after the preparation to which is added is 100% is shown.

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 can be seen that it has the same CK activation ability as C. Further, when added to an aqueous solution, it is possible to retain the CK activation ability for a long period of time, among the various SH compounds including NAC, TG, 2M which is the CK activator according to the present invention.
It turns out that it is only E and 2MES. Furthermore, from the results in Table 4, when NAC and TG were used in combination as CK activators, CK after storage for 1 month at both 10 ° C and 20 ° C.
Decrease in activation capacity, and when NAC and 2ME are used in combination, at 10 ° C, C
Although the K activation ability is stable, it can be seen that the CK activation ability after storage for 1 month at 20 ° C. is obviously decreased. From these, it is understood that it is difficult to prepare a liquid reagent for measuring CK activity that is stable for a long period of time according to the conventional idea that NAC is used as a main component of the CK activator. Further, considering the results of Reference Example 1, even if the oxidation product does not inhibit CK activity, C
Unsuitable as a CK activator for a liquid reagent for K activity measurement, that is, a compound such as TDG or DTT that cannot retain CK activation ability in a solution for a long period of time, for example, AHMP. It can be understood that there are some such as 2-thiouracil and the like which do not show sufficient CK activation ability as a result from the problem of solubility and the like. From these, as a CK activator for a liquid reagent which requires storage stability for a long period of time, it has at least a sufficient CK activation ability, and a CK stable for a long period of time in an aqueous solution. It is considered that the TG, 2ME and 2MES according to the present invention are required to have properties such as being able to retain the activation ability, and further, the oxidation product thereof does not inhibit CK activity. It is understood that all of these conditions are satisfied.

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

Comparative Example 2. [Enzyme Solution] The same reagents as in Example 1 were used except that 26 mM of NAC was used instead of the CK activator used in Example 1. What was prepared by using was used. [Substrate solution] Same as in Example 1. [Sample] Same as in 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 performed by the same operation as in Example 1 except that the above sample was used. Ten
Table 5 shows the average value of each CK activity value of each type of sample.

[0039]

[Table 5] * Figures in parentheses show relative values when the enzyme solution prepared at the time of use (containing 100 mM of TG as a CK activator or 26 mM of NAC) and the average value of CK activity measured using the substrate solution are 100%. Show.

As is clear from the results shown in Table 5, the conventional CK activity measuring reagent using NAC as the CK activator showed a marked decrease in the CK activity measured value 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 has no effect on the measured CK activity value even after storage at 10 ° C. for 7 months, and it can be seen that it retains the performance immediately after preparation. . Further, as a result of studying the calibration range, the liquid reagent for CK activity measurement 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 performance immediately after preparation. I confirmed that I was holding it.

Example 3 The following is a typical example of a liquid reagent kit that can be stored for a long period of time and is used to measure CK activity in body fluids such as serum. (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 liquid reagents and a liquid reagent for measuring CK activity for measuring CK activity in body fluids such as serum using the activator. The CK activator of the present invention is stable for a long period of time and
When it is used as a CK activator for liquid reagents, it does not produce an oxidation product having an activity-inhibiting effect and can be stored for a long time, and at least 3 when stored at low temperature, for example, 10 ° C. It has a remarkable effect that it can be used for a month, usually 6 months or more without deterioration in performance.

Claims (6)

[Claims] 1. A creatine kinase activator for a liquid reagent containing at least one compound selected from the group consisting of thioglycerol, 2-mercaptoethanol, and 2-mercaptoethanesulfonic acid or a salt thereof. 2. A method for measuring creatine kinase activity, which comprises at least one compound selected from the group consisting of thioglycerol, 2-mercaptoethanol, and 2-mercaptoethanesulfonic acid or a salt thereof. Liquid reagent. 3. 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 containing at least one compound selected from the group consisting of sulfonic acids or salts thereof and a pH of 7.5 containing creatine phosphate.
To 10 second reagent, wherein each of glucose and magnesium ion is contained in at least one of the first reagent and the second reagent, and a liquid reagent for measuring creatine kinase activity. 4. Glucose and magnesium ions are first
The liquid reagent for measuring creatine kinase activity according to claim 3, which is contained in the reagent. 5. The liquid reagent for measuring creatine kinase activity according to claim 3, wherein the magnesium ion is derived from magnesium acetate and / or magnesium chloride. 6. Imidazole-acetic acid 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 pH comprising glucose and creatine phosphate,
A liquid reagent kit for measuring creatine kinase activity, which is characterized by being combined with 7.5 to 10 second reagents.