JP3045190B2 - Creatine kinase measurement reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring creatine kinase (hereinafter abbreviated as CK) in a biological fluid, which is useful for medical diagnosis and other purposes.

[0002]

2. Description of the Related Art CK is present in muscle tissue and brain throughout the body,
In the field of clinical tests, CK activity is one of the important items routinely measured for the diagnosis of heart diseases such as myocardial infarction, muscular diseases such as muscular dystrophy, neurological diseases, central nervous system diseases, and psychosis. is there. CK is an enzyme that catalyzes the reaction in both left and right directions in the following reaction formula 1.

[0003]

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Conventionally, various methods for measuring CK activity have been proposed, but the method for measuring the reaction in the leftward direction has hardly been used in recent years because of low sensitivity and unstable coloring. In addition, the method for measuring the activity in the right direction includes a method of reacting the produced creatine with a dye to perform colorimetry, a method of measuring fluorescence, and a method of using luciferase (Japanese Patent Application Laid-Open No. SHO 51-51).
-41597, JP-B-58-5678, JP-A-56-26200, JP-A-57-10519
No. 9), a method using phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (JP-B-59-34119, JP-A-56-155).
No. 00), a method using hexokinase (hereinafter abbreviated as HK) and glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH).

[0005] Among them, the method using HK and G6PDH is most frequently used because it is excellent in principle, has good sensitivity and reproducibility, and can process a large number of samples. Further, in the method using HK and G6PDH, there was a problem that storage stability at room temperature in a solution state was poor. However, instead of HK, glucoside having extremely high specificity to glucose and having excellent stability was used. A method using a kinase (hereinafter abbreviated as GlcK) has been proposed (JP-A-56-169598, JP-A-59-1).
No. 51899, JP-A-61-88899, JP-A-61-289900, JP-A-62-1045
No. 98). The method using GlcK and G6PDH is a method in which storage stability and accuracy of measurement are further added to the method using HK and G6PDH, which are frequently used because they are excellent in principle and have good sensitivity and reproducibility.

[0006]

However, these conventional reagents for measuring CK have a comparatively narrow measurement range, and can be used for measurement when serum CK is rapidly increased due to myocardial infarction or muscle disease after exercise. It was often recognized that measurement was not possible without dilution each time. For this reason, it is unsuitable when an urgent test is required due to myocardial infarction or the like, and the measurement is complicated, causing a measurement error.

[0007] As described above, the present invention provides a CK having a measurement range in which even a sample containing CK at a high concentration can be quantified without dilution.
It is intended to provide a reagent for measurement.

[0008]

Means for Solving the Problems The present inventors have conducted various studies with the aim of providing a reagent for measuring CK that satisfies the above requirements. As a result, 6-phosphogluconolactonase was used as a reagent for measuring CK. In order to achieve the above object, the CK measurement reagent according to the present invention has been found to be a measurement reagent having sufficient performance for daily use, and to complete the present invention. Was.

That is, the present invention provides creatine phosphate, adenisine 5'-diphosphate (hereinafter abbreviated as ADP), glucose, HK or GlcK, nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or A CK measurement reagent containing nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP), G6PDH, magnesium salts, and SH reagent as main components, for CK measurement containing 6-phosphogluconolactonase. It is a summary of the reagent.

Hereinafter, the present invention will be described in detail. The reagent of the present invention comprises, in addition to 6-phosphogluconolactonase, creatine phosphate, ADP, glucose, HK or Glc.
In addition to the main components of K, NAD or NADP, G6PDH, magnesium salts, and SH reagent, additives and buffers such as ordinary activators are contained.

In the present invention, it is necessary to use 6-phosphogluconolactonase.
-Phosphogluconolactonase is an enzyme that hydrolyzes 6-phosphogluconolactone to 6-phosphogluconic acid, which is classified under the classification number EC 3.1.1.13 by the International Commission on Enzymes of the Biochemical Union. is there. The source is not particularly limited, and those derived from animals, plants, and microorganisms can be used without any problem. Microorganism-derived ones are preferred because of their easy handling and availability. These include Zymomonas sp., Escherichia coli, and Leuconostoc (Le
uconostoc), Lactobacillus
Genus, Pseudomonas, Bacillus (Ba
There are enzymes from various sources, including the genus cillus. Among them,
Those belonging to bacteria belonging to the genus Zymomonas, such as Zymomonas mobilis and Zymomonas anaerobia, are preferred. Specifically, Zymomonas mobilis ATCC 31821 strain, 31
823 shares, 35000 shares, 35001 shares, 10988 shares
Strains, 29191 strains, 29129 strains and the like.

The above microorganism is cultured in a commonly used medium, and the 6-phosphogluconolactonase used in the present invention is isolated from the obtained cells by a commonly used separation and purification method. Can be obtained.

The source of the HK used in the present invention is not limited, and HKs of various origins such as those derived from microorganisms and those derived from animals can be used. Among them, those derived from microorganisms such as baker's yeast can be used easily. GlcK, which has a higher substrate specificity for glucose than HK, can also be used. Gl
The source of cK is not limited, and those of various origins such as those derived from microorganisms and those derived from animals can be used. Among them, genus Bacillus, Thermoactinomyces, Thermos (Thermus) because of their ease of handling and availability.
Microorganisms such as genus, Thermomicrobium and Zymomonas are preferred, and particularly preferred microorganisms include Bacillus stearothermophilus and Zymomonas mobilis. it can.

The source of G6PDH is not limited, for example, from microbial origin such as yeast, animal origin, etc., but preferably not only NADP but also NADP as a coenzyme
G6PDH, which also acts on D, is preferred. Of these, G6PDH of microbial origin is preferred, and specifically, Leuconostoc mesenteroides.
Genus Leuconostoc, Pseudomonas fluorescens
) And Zymomonas genus such as Zymomonas mobilis. More preferably, both NADP and NAD can be used as coenzymes,
G6PD derived from a thermophilic bacterium which is rich in stability and preservation
H is desirable.

As the magnesium salts, for example, salts such as magnesium acetate and magnesium sulfate can be used without any trouble.

Examples of SH protective agents include N-acetylcysteine (hereinafter abbreviated as NAC), dithiothreitol, dithioerythritol, mercaptoethanol, thioglycerol, thioglycolic acid, thioglucose, and cysteine. NAC that is easy to handle and easy to handle is preferable. As the preservative, for example, a known agent such as sodium azide can be used without any trouble.

As the stabilizer, for example, soluble starch,
Polysaccharides and derivatives thereof such as methylcellulose and carboxymethylcellulose, proteins such as albumin and γ-globulin, and water-soluble polymer compounds such as polyvinyl alcohol and polyethylene glycol can be used as appropriate.

As for creatine phosphate, ADP, glucose, NAD or NADP, those used in conventional reagents for measuring creatine kinase can also be used.

In general, the concentrations of the components of the reagent for measuring CK of the present invention are preferably as follows. For example, HK or G
lcK is 0.1 to 40 units / ml, G6PDH is 0.1 to 40 units / ml, 6-phosphogluconolactonase is 0.01 to 20 units / ml, creatine phosphate is 2 to 70 mM, and ADP is 0. . 1-20 mM, N
AD or NADP is 0.05 to 20 mM, glucose is 1 to 200 mM, and magnesium salts are 0.5 to 30 m.
M, SH reagent 0.1 to 100 mM, adenosine 5'-
Monophosphate (hereinafter abbreviated as AMP) 0.2 to 20 m
M, 1 to 100 μM of diadenosine pentaphosphate (hereinafter abbreviated as Ap5A), 0.1 to 20 mM of ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), and 0.5 to 50 mM of sodium azide. More preferably, HK or GlcK is 0.2 to 20 units / ml, and G6PDH is 0.2 to 20 units / ml, 6
0.05 to 15 units / ml of phosphogluconolactonase, 5 to 40 mM of creatine phosphate, 0.2 to 10 mM of ADP, 0.1 to 10 of NAD or NADP.
mM, glucose 2-100 mM, magnesium salts 2-15 mM, SH reagent 0.5-50 mM, AMP
0.5 to 15 mM, Ap5A 2 to 50 μM, EDT
A: 0.2 to 10 mM, sodium azide: 1 to 30 m
M may be used.

The measuring reagent of the present invention can be used in a so-called one-reagent system in which all components are converted into one reagent, but depending on the convenience of an automatic analyzer or the like, a so-called two-reagent system is used. It can also be used.

The principle of measurement of the reagent for measuring CK of the present invention is as follows. ATP produced by the reaction in the rightward direction of Reaction Formula 1 is reacted with GlcK in the presence of 6-phosphogluconolactonase as shown in the following Reaction Formulas 2 and 3. And G6PDH using a conjugation effect.

[0022]

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[0023]

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That is, the increase in absorption at 340 nm of NAD (P) H produced by the conjugation reaction of Reaction Formulas 1 to 3 is measured to quantify the CK activity in the sample.

In order to measure CK activity in a sample using the CK measurement reagent of the present invention, for example, a measurement reagent is placed in a spectrophotometer cell in which the cell chamber is kept warm, and the measurement temperature (20
(Arbitrary temperature between -45 ° C) for about 3 minutes, then add the sample, mix and measure the increase in absorbance at 340 nm. In addition, as the reaction time of the measurement after the addition of the sample, an arbitrary time can be selected after 30 seconds, but in the case of an automatic analyzer, conditions suitable for the measurement conditions of the apparatus are set. I just need.

[0026]

Next, the present invention will be described specifically with reference to examples. Example 1, Comparative Example 1 6-phosphogluconolactonase was prepared using FEBS Letters, 193, 185-188, 198.
The cells were cultured and isolated and purified according to the method described in 5 years. That is, 15% glucose, 0.5% yeast extract,
1 mg of biotin per liter of a solution containing 0.05% of monopotassium phosphate and 0.05% of magnesium sulfate;
mg of calcium pantothenate and 20 mg of ammonium ferrous sulfate (hexahydrate) in a medium containing Zymomonas mobilis ATCC 31821.
The strain was cultured.

After culturing, the collected cells are crushed, and green H
E-4BD-Sepharose column chromatography (Green HE-4BD is purchased from ICI Japan as Procion Green HE-4BD), eluted with a buffer containing G6P, and then Cellulofine GCL-2000 column Purified using chromatography (purchased from Seikagaku Corporation).

A reagent for measuring CK was prepared using 6-phosphogluconolactonase purified as described above. Bacillus stearothermop
hilus) [purchased from Seikagaku Corporation]
3 units / ml, G6PDH from Leuconostoc mesenteroides
[Purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] 1
Units / ml, 6-phosphogluconolactonase
4 units / ml, ADP [purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] 2 mM, NADP [purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] 2 mM, glucose 20 mM, AMP [purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] 5 mM, Ap5A [Boehringer
Purchased from Mannheim Yamanouchi Co., Ltd.] 10 μM, NAC2
0 mM, magnesium sulfate 10 mM, sodium azide 0.1%, EDTA 2 mM, creatine phosphate 25 mM
80m containing imidazole-acetate buffer (pH 6.7)
A reagent for measuring CK consisting of M was prepared (Example 1). Separately, a reagent for measuring CK from which 6-phosphogluconolactonase was removed from Example 1 was prepared (Comparative Example 1).

Next, CK derived from rabbit muscle was used as a sample.
[Purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] was dissolved in physiological saline or commercially available standard serum to prepare a sample. 3.0 ml of the reagent for CK measurement prepared above was placed in a cell having an optical path length of 1 cm, kept at 37 ° C. for about 3 minutes, and 0.06 ml of the sample prepared above was added to start the reaction.
The change in absorbance at 40 nm was followed. Similar measurements were performed on samples containing various concentrations of CK, and the relationship between the change in absorbance per minute and the CK activity in the sample was shown in FIG. From this figure, it can be seen that in Example 1, the amount of change in absorbance per minute was about 4% for CK.
While it changed linearly up to 2,000 units / l, in Comparative Example 1, it was found that it changed linearly only up to about 2000 units / l. As described above, it was shown that the range of measurement was greatly expanded by including 6-phosphogluconolactonase.

Example 2 GlcK and G6PDH were purified from the Biochemical Journal, Vol.
The culture was performed according to the method described on page 634, 1985, and each was isolated and purified. That is, 15% glucose,
One liter of a solution containing 0.5% yeast extract, 0.05% monopotassium phosphate, 0.05% magnesium sulfate contains 1 mg of biotin, 2 mg of calcium pantothenate and 20 mg of ammonium ferrous sulfate (hexahydrate ) And a medium containing Zymomonas mobili
s) The ATCC 29191 strain was cultured.

After culturing, the collected cells are disrupted, and then subjected to Scarlet MX-G-Sepharose chromatography (Scarlet MX-G is a trade name of Procion Scarlet M.
X-G) (purchased from ICI Japan)
The lcK was eluted with an increase in pH and the G6PDH was eluted with the addition of NADP, followed by ammonium sulfate fractionation. Further, both enzymes were subjected to gel filtration by Sephacryl S-200 chromatography to obtain a purified enzyme.

GlcK and G6PDH purified as described above
Was used at the same concentration as in Example 1 to prepare a CK measurement reagent (Example 2). Separately, a reagent was prepared by removing 6-phosphogluconolactonase from Example 2 (Comparative Example 2). When the same experiment as in Example 1 was performed using these reagents, in Example 2, the change in absorbance per minute was in a linear relationship up to the amount of CK in the sample of about 4000 units / l. Showed a linear relationship only up to about 2000 units / l.

Example 3 Example 1 or Example 2 except that the concentration of 6-phosphogluconolactonase was changed to 0.05 unit / ml and 15 units / ml in the reagent of Example 1 or Example 2. Two kinds of reagents each having the same composition as the above reagent were prepared. Using these reagents, the relationship between the amount of change in absorbance per minute and the amount of CK in the sample was examined. As a result, a linear relationship of up to about 4000 units / l of CK was observed for all four reagents. It was found to show a range.

Example 4, Comparative Example 3 Instead of GlcK, yeast-derived HK [purchased from Boehringer Mannheim Yamanouchi Co., Ltd.] 3.5 units / ml
And the same reagent as in Example 1 and Comparative Example 1 except for using 1.5 units / ml of yeast-derived G6PDH (Example 4, Comparative Example 3). When the same experiment as in Example 1 and Comparative Example 1 was performed, the linear relationship between the amount of change in absorbance per minute and the amount of CK in the sample was up to about 3500 units / l in Example 4, and about 1500 in Comparative Example 3. Units / l were obtained.

Example 5 Bacillus stearot
hermophilus) GlcK 3.75 units / ml,
Leuconostoc me
senteroides) G6PDH 1.25 units / m
1, 0.5 unit / ml of 6-phosphogluconolactonase obtained in Example 1, 2.5 mM of ADP, 2.5 mm of NADP.
5 mM, glucose 25 mM, AMP 6.25 mM,
Reagent 1 comprising 100 mM imidazole-acetate buffer (pH 6.7) containing 12.5 μM of Ap5A, 25 mM of NAC, 0.1% of sodium azide and 2.5 mM of EDTA
And creatine phosphate 125 mM, magnesium acetate 5
Reagent 2 consisting of 0 mM was prepared.

[0036] Reagent 1, 0.4 ml prepared above was added to 37
5 minutes after adding 0.01 ml of the sample prepared in Example 1 to the cell having an optical path length kept at 1 ° C., reagent 5 was added, and the change in absorbance at 340 nm was followed for about 5 minutes (automatic analyzer). Hitachi 7050 type). It was found that the change in absorbance per minute and the amount of CK in the sample had a linear relationship up to about 4000 units / l of CK.

[0037]

According to the present invention, the reagent for measuring CK of the present invention is
For conjugating G6PDH with G6PDH, GlcK and G6PDH
The range of measurement, which was a problem of the method of conjugating
This is greatly expanded by using phosphogluconolactonase. It has become a highly reliable measurement reagent with sufficient performance to routinely analyze and quantify CK in biological fluids of patients with myocardial infarction and other muscular diseases, which have been increasing in recent years. As described above, the reagent for measuring CK of the present invention has made a great contribution to the field of clinical examination.

[Brief description of the drawings]

FIG. 1 shows the amount of CK (unit / l) in a sample when a creatine kinase measuring reagent of the present invention (Example 1) and a conventional measuring reagent (Comparative Example 1) were used, and the amount of CK per minute at 340 nm. It is explanatory drawing which shows the absorbance change amount.

[Explanation of symbols]

 ● Example 1 ○○ Comparative Example 1

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/25-1/66 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. creatine phosphate, adenosine 5'-diphosphate, glucose, hexokinase or glucokinase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, glucose-
A creatine kinase measuring reagent comprising 6-phosphate dehydrogenase, magnesium salts, and an SH reagent as main components, which comprises 6-phosphogluconolactonase.