JPS5995900A - Composition for determination - Google Patents

Abstract

PURPOSE:To provide a composition for the determination of ATP, etc., storable for a long period at room temperature, effective with a small amount of enzyme, and having excellent economicity, by adding a hydrophilic organic solvent to glycokinase or hexokinase and glucose-6-phosphate dehydrogenase. CONSTITUTION:The objective composition is prepared by adding (A) preferably 1-30wt% of a hydrophilic organic solvent such as dimethyl sulfoxide to (B) glycokinase or hexokinase and (C) glucose-6-phosphate dehydrogenase. The components (B) and (C) are preferably those produced by microorganisms having optimum growth temperature of 50-85 deg.C. USE:Determination of glucose and creatine phosphokinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for measuring glucose, ATP, creatine phosphokinase (hereinafter referred to as CPK), various other phosphotransnucleases, and various glycosidases that release glucose. .

Conventionally, hexokinase (hereinafter referred to as HK) or glucokinase (hereinafter referred to as GK) and glucose-6-
A method using phosphoric acid dehydrogenase (hereinafter referred to as 06PDH) as a coupled enzyme, the so-called HK/G6PDH system or GK/G6PDH system, is used in the field of clinical testing to measure glucose and CPK.

What is the measurement principle?

CPK CP+ADP--→C+ATP (
1) ATP + Gu μ course - Giant] Bu vassal master 9mu AI) p
10, G6p (2) G6P + NAD (Pl danting凋L6-PGA + NADu) + u (3)

(The above abbreviations are CP: creatine phosphate, C: creatine, ADP: adenosine-51-diphosphate, AT
P: adenosine-5'-triphosphate, 06P nigercose-6-phosphate, NAD(P)H: reduced niconanamide adenine dinucleotide (phosphate), '6
-PGA: represents 6-phosphogluconic acid. ) The enzymes that catalyze the above reaction formulas (1), (2), and (3) are CPK, HK, GK, and 06PDH, respectively.

However, conventional compositions for measuring glucose and CPK based on the HK/G6PDH system have poor storage stability at room temperature in a solution state, and the reagents have low storage stability at room temperature (1
The lifespan at temperatures between 8 and 35°C was very short. For this reason, JP-A-56-124397 and JP-A-56
-169598 publication, heat resistant GK and heat resistant 06
A quantitative composition using PDH has been proposed. Although the room temperature storage stability of this composition has been improved, it is still not sufficient due to the instability of the coupling enzyme and the instability of the SH compound contained in the composition, and it is difficult to maintain stability for a long period of time. required a relatively large amount of enzyme.

From this point of view, the present inventors conducted extensive research with the aim of providing a measurement composition that can be stored for a long time at room temperature, is effective with a small amount of enzyme, and is highly economical. The inventors have discovered that all of the above objects can be achieved by using a hydrophilic organic solvent, and have completed the present invention.

That is, one aspect of the present invention is a measurement composition comprising glucokinase or hexokinase and glucose-6-phosphate dehydrogenase, which is characterized by containing a hydrophilic organic solvent.

Various types of glucokinase, hexokinase, and glucose-6-phosphate dehydrogenation ffI used in the present invention can be used, including those derived from microorganisms and those derived from animals. Those produced by microorganisms at a temperature of 50°C to 85°C are preferred from the viewpoint of stability. Such microorganisms include, for example, Bacillus stearothermophilus, Bacillus suthermoproteolii, Bacillus acidocaldarius, Thermoactinomyces sp., Thermus sp., Thermomicrobilum sp., Calburia sp. Examples include microorganisms. Among these, a particularly preferred microorganism is Bacillus stearothermophilus.
hermophilus) is raised l Sufu 70
As a specific example of 'l---1 = office,
ATCC795! l.

7954, 8005. 10194. 12980.
Examples include NOA 1503.

The hydrophilic organic solvent used in the present invention includes:

Any organic solvent may be used as long as it is easily mixed with water. Such substances include polyhydric alcohols such as methyl alcohol, ethyl alcohol, natonohydric alcohols, ethylene glycol, and glycerin;
Amides such as N-N-monomethylformamide and N-N-dimethylformamide, acetone, n
Examples include ketones such as -butyl ketone, ethers such as dioxane and tetrahydrofuran, enethers such as methyl acid and ethyl acetate, and dimethyl nulphoxide, which can be used alone or in combination.

This hydrophilic organic solvent is added to the composition in an amount of 0.1 to 50% by weight, preferably 1 to 60% by weight, and more preferably 3 to 50% by weight.
It is sufficient to use 20% by weight.

Next, to prepare the composition of the invention, glucose,
It is suitably prepared according to the type of target substance to be measured, such as CPK, ATP, and other biological components.

ADP or its salts, ATP or its salts, glucose, NAD or NADP or its salts, salts containing magnesium, GK or HK, G6PDH and tris-me, imidazole-acetic acid, triethanolamine-hydrochloric acid, etc. buffers (pI 45~ 10) etc. may be added. In addition, suitable amounts of thio μ compounds, chelating reagents, specific reaction inhibitors, preservatives, surfactants, stabilizers, etc. can be added as necessary. These may be blended in any order. Further, the usage amount of these may be 9, for example, the following amount.

GK or HK i O, 1-20 nits/
d06PDHi O, 1~20;
Phosphorophore fuses to be measured,
0-40mM substrate (phosphate donor) Glycolymse substrate to be measured; 0-4
0mMADP or its salt +0 to 20mMATP
or its salt; 0-20mM 0mM glucose
0-40mM NAD or NADP or its salt 1 1-10mM buffer containing 0.1-10mM magnesium (pH 5-10)
+ 10 to 500 n+M As mentioned above, the composition of the present invention can measure glucose and CPK, which are important measurement items in the field of clinical testing.

It can be applied not only to the measurement of ATP, etc., but also to the measurement of the activity of honuhotrasysphaefuse or glycosidase that releases glucose.

In the composition of the present invention, since the WA aqueous organic solvent activates the coupled enzyme reaction, the amount of enzyme used in the measurement reagent can be reduced, and the room temperature stability of the measurement reagent in the solution state can be increased. I was able to prepare a lot of reagents at once, so I used a large amount of reagents at once! In addition to improving work efficiency, it is also possible to reduce the frequency of discarding surplus reagents.

Next, the present invention will be specifically explained using examples.

Example 1. Comparative Example 1 Heat-resistant GK2 unit)/d obtained from Bacillus stearothermophilus NCA-1503, heat-resistant 06PDH2 unit obtained from the same bacterial cell/TsujiNADP
2mM, ATP2mM, MgCIQ2
A composition for measuring glucose was prepared so as to have a concentration of 10% by weight of dimethyl sulfoxide and 100 mM of Trinu hydrochloride buffer (pH 8.5).

Glucose was measured using this composition using a standard serum with a known glucose concentration as a test solution using an end point method.

The standard serum used was one in which the concentration of GIV''2-s in the standard serum was 2001 P/dd.

As a measurement method, the above composition was kept at 30°C.

0.6 ml of the solution was put into a cell with an optical path length of 1aI, and the absorbance (AO) of 340 dishes was measured using a spectrophotometer whose cell chamber was kept at a constant temperature of 50°C.

Next, 4 μl of glucose measurement was added to the cell, the enzyme reaction was allowed to proceed, and the absorbance (A) at 540 nm was measured 10 minutes later.

The glucose concentration in the sample was calculated using the following formula.

Glucose concentration (da/d4) -437 (A Ao
) Also, the above composition was stored in a constant temperature tV at 30°C, and after 7 days, the stored composition was used to measure glucose in the above standard serum! l! provided.

For comparison, the same procedure as in Example 1 was carried out except that dimethyl sulfoxide was not added.

The results are shown in Table 1. In Example 1, a
The same value was obtained both on the day of storage and after 7 days of storage at 30°C, indicating the stabilizing effect of the present invention.

Example 2. Comparative Example 2 The GK and 06FDH concentrations of Example 1 were reduced to one-fifth,
The same procedure as in Example 1 was carried out except for this.

Also, for comparison, without adding dimethyl sulfoxide,
The rest was carried out in the same manner as above.

The results are shown in Table 1.

From Table 1, it is clear that in Example 2, almost the same measured values as in Example 1 were obtained, making it possible to reduce the enzyme concentration.

Example 3. Comparative Example 6 GK of Example 1 was replaced with HK obtained from yeast, heat-resistant 06
The same procedure as in Example 1 was carried out except that PDH was changed to 06FDH obtained from lactic acid bacteria.

Also, for comparison, without adding dimethyl sulfoxide,
The rest was the same as above.

The results are shown in Table 1f.

From Table 1, the stabilizing effect of the present invention is clear also in the HK/G6PDH system.

Table 1 Examples 4-51 Comparative Examples 4-5 Heat-resistant GK3-L chive used in Example 1) / w5
G6PDH3 is also heat resistant! :, t/ #/ ,
ADP ImM.

Glucose 12mM, NADP 1.6mM
, magnesium acetate 5mM, AMP 4mM,
Dithiothreitol 10mM.

Sodium azide 10mM, creatine phosphate 20mM
A composition for measuring CPK (Example 4) was prepared, consisting of 100 mM glycerin and 100 mM imidazole-acetic acid buffer (pH 6,7).

Further, for comparison, a composition (Comparative Example 4) was prepared in the same manner as above except that glycerin was not added. Furthermore, the measurement composition (
Example 5) was produced. Further, for comparison, a composition (Comparative Example 5) was prepared in the same manner as in Example 5 except that glycerin was not added.

Next, each of the above compositions was kept warm at 30°C, and 0.6 ml of it was put into a cell with an optical path length of 11.
CP in the sample was determined by the change in absorbance at 340 nm using a spectrophotometer with the cell chamber kept at a constant temperature of 60°C.
Measurement of K activity was performed.

Next, each of the above compositions was stored in a constant temperature bath at 60°C for 3 days.
After 5 and 7 days, these compositions and fresh t
The composition of Example 4 thus prepared was used.

The activity of CPK in standard serum containing CPK was measured. The measured value of the composition of Example 4 newly prepared on each day was 1004 J, and the composition t stored at 30°C
FIG. 1 shows the relative ratio of these measured values. 1 in the figure is Example 4, 2 is Example 5, and 6 is Comparative Example 4, 4.
shows Comparative Example 5.

It is clear from FIG. 1 that the composition of the present invention is stable over a long period of time.

[Brief explanation of drawings]

Figure 1 shows that the CPK activity of standard serum was measured using various CPK measurement compositions after the number of storage days shown on the horizontal axis t, and on the day of measurement, the measurement composition of Example 4, which was newly prepared, was used. The vertical axis shows the relative ratio of the obtained standard serum to the CPK activity value, where 1 indicates Example 4 and 2 indicates Example 5.
, 6 indicates Comparative Example 4, and 4 indicates Comparative Example 5. Patent applicant Unitika Co., Ltd.

Claims (4)

[Claims] (1) A measurement composition comprising glucokinase or hexokinase and glucose-6-phosphate dehydrogenase, which contains a hydrophilic organic solvent. (2) The measurement composition according to claim 1, wherein the glucokinase is produced by a microorganism whose optimal growth temperature is 50°C to 85°C. (3) The measuring composition according to claim 1, wherein the hexokinase is a hexokinase produced by a microorganism whose optimal growth temperature is 50°C to 85°C. (4) The glucose-6-phosphate dehydrogenase is glucose-6-phosphate dehydrogenase produced by a microorganism whose optimal growth temperature is 50°C to 85°C. Composition for measurement.