Reagent for assaying creatine kinase

A reagent system for assaying creatine kinase is disclosed. The reagent system consisting essentially of a first reagent comprising glucose-6-phosphate dehydrogenase, .beta.-nicotinamideadenine dinucleotide (phosphate), and adenosine diphosphate, and a second reagent comprising creatine phosphate, said second reagent being maintained at a pH of from 7.5 to 10, and at least one of said first reagent and said second reagent containing glucokinase and glucose. The creatine kinase-assaying reagent exhibits remarkably improved stability in a dissolved state so that it can be prepared in large quantities and can be conventionally utilized to cope with urgent clinical examinations.

FIELD OF THE INVENTION 
This invention relates to a reagent for assaying creatine kinase in body 
fluids, such as blood serum, urine, and the like. 
BACKGROUND OF THE INVENTION 
Creatine kinase (E.C. 2.7.3.2 registered in International Union of 
Biochemistry) is an enzyme present in muscular tissues throughout the body 
and in the brain. In the field of clinical examinations, an assay of 
creatine kinase activity is one of the important examinations usually 
carried out for diagnosis of cardiac diseases, e.g., myocardial 
infarction, muscular diseases, e.g., progressive muscular aystrophy, 
nervous diseases, central nervous system diseases, mental disorders, and 
the like. 
Creatine kinase is an enzyme which catalyzes the reversible reaction shown 
by the following scheme (1) in both directions: 
##STR1## 
wherein ADP is adenosine diphosphate; and ATP is adenosine triphosphate. 
Various methods have conventionally been proposed for assaying creatine 
kinase. One type of method comprises assaying the catalytic activity in 
the direction to the left of the above-described reaction (1). This type 
of method includes (a) a method of measuring an inorganic phosphoric acid 
released by hydrolysis of creatine phosphate, (b) a method comprising 
converting ADP to the oxidation of reduced from .beta.-nicotinamideadenine 
dinucleotide (hereinafter abbreviated as NADII) by the action of pyruvate 
kinase and lactate dehydrogenase and measuring the decrease in absorption 
at 340 nm due to the oxidation of NADH, (c) a method comprising converting 
ADP to pyruvic acid by the action of pyruvate kinase and measuring 
hydrazone produced by the reaction between pyruvic acid and 
2,4-dinitrophenylhydrazine, and the like. In recent years, however, 
scarcely has any of those methods been employed, due to their low 
sensitivity or unstable color development. On the other hand, a method for 
assaying the activity of creatine kinase in the direction to the right of 
the above-described reaction (1) includes (d) a colorimetric or 
fluorometric method in which creatine produced is reacted with a dye, (c) 
a method of using luciferase as disclosed in Japanese Patent Application 
(OPI) Nos. 41597/76, 26200/81 and 105199/82 (the term "OPI" herein used 
means "unexamined published application") and Japanese Patent Publication 
No. 5678/83, (f) a method of using phosphoglycrerate kinase and 
glyceraldehyde-3-phosphate dehydrogenase as disclosed in Japanese Patent 
Publication No. 34119/84 and Japanese Patent Application (OPI) No. 
155000/81, (g) a method of using hexokinase and glucose-6-phosphate 
dehydrogenase, and the like. Of these, the colorimetric or fluorometric 
method (d) has poor reliability on the measured values; the luciferase 
method (e) requires expensive luciferase and a specific apparatus for 
measurement; and the phosphoglycerate kinase/glyceraldehyde-3-phosphate 
dehydrogenase method (f) is an absorption decreasing system similar to the 
pyruvate kinase/lactate dehydrogenase method as described above, and, 
therefore, involves the same disadvantages as associated with the pyruvate 
kinase/lactate dehydrogenase method and, in addition, requires use of 
phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase that 
are more expensive than pyruvate kinase and lactate dehydrogenase. Hence 
any of the methods (d) to (f) is not satisfactory for practical use. The 
hexokinase/glucose-6-phosphate dehydrogenase method (g) has been employed 
most commonly because it is based on the most reasonable principle, 
exhibits satisfactory sensitivity and reproducibility and is capable of 
assaying a number of specimens. The principle of this assay method 
consists in the absorption increase at 340 nm due to the formation of 
reduced form .beta.-nicotinamideadenine dinucleotide (phosphate) which is 
finally produced by the following reaction schemes: 
##STR2## 
wherein NAD(P) is .beta.-nicotinamideadenic dinucleotide (phosphate); and 
NAD(P)H is reduced form .beta.-nicotinamideadenine dinucleotide 
(phosphate). 
Ever since the first report on the hexokinase/glucose-6-phosphate 
dehydrogenase method by T.T. Oliver in Biochem. J., Vol. 61, pp. 116-122 
(1955), various improvements have been made. For example, there have been 
conducted studies on an assay method for inhibiting an activity of 
adenylic kinase that mainly exists in blood and causes a negative error in 
the hexokinase/glucose-6-phosphate dehydrogenase method, as described in 
U.S. Pat. No. 4,220,714, European Pat. No. 71087 (corresponding to 
Canadian Pat. No. 1,175,737), G. Szasz, W. Gerhardt, W. Gruber and E. 
Bernt, Clin. Chem., Vol. 22, pp. 1806-1811 (1976) and G. Szasz, W. 
Gerhardt and W. Gruber, Clin. Chem., Vol. 23, pp. 1888-1892 (1977); 
studies on thiol compounds for activation of creatine kinase as described 
in Japanese Patent Application (OPI) No. 106397/74 (DE 2302721) and G. 
Szasz, W. Gerhardt and W. Gruber, Clin. Chem., Vol. 24, pp. 1557-1563 
(1978); studies on the use of chelate compounds and stability of a reagent 
for assaying creatine kinase activity as described in G. Szasz, J. 
Waldenstrom, and W. Gruber, Clin. Chem., Vol. 25, pp. 446-452 (1979); and 
the like. As a result, the hexokinase/glucose-6-phosphate dehydrogenase 
method has been established as the most reliable assay method for creatine 
kinase in clinical laboratories. 
Nevertheless, the hexokinase/glucose-6-phosphate dehydrogenase method still 
has problems awaiting solution with respect to analytical accuracy and 
stability of a reagent for assaying creatine kinase. The former problem 
comes from the possible action of hexokinase on sugars other than glucose 
existing in body fluids, such as fructose and mannose, which results in a 
positive error of measured values. The latter problem is ascribed to the 
poor stability of the reagent during preservation in the form of a 
solution, i.e., a short working life of the reagent in a liquid state at 
room temperature (18.degree. to 35.degree. C.), even if a stabilizer, such 
as phycol (as described in Japanese Patent Application (OPI) No. 12897/77) 
and albumin, is added. Moreover, even in a so-called two-reagent system 
wherein the reagent is divided into two containers in a pH region of from 
6.5 to 7.0, the stability of the reagent cannot be improved as desired so 
as to withstand use for a prolonged period of time in clinical 
laboratories. Therefore, satisfactory solutions to these problems have 
been strongly desired. 
In order to overcome the above-described disadvantages encountered in the 
hexokinase/glucose-6-phosphate dehydrogenase method, a one-reagent system 
using glucokinase having extreme specificity to glucose and excellent heat 
stability has been described to be used in place of hexokinase, as 
disclosed in U.S. Pat. No. 4,438,199 (corresponding to European Patent 
Publication No. 43181A and Japanese patent application (OPI) No. 
169598/81) and U.S. patent application Ser. No. 580,503 (corresponding to 
European Patent Publication No. 119722A and Japanese Patent Application 
(OPI) No. 151899/84). This glucokinase/glucose-6-phosphate dehydrogenase 
method also improves stability of a reagent for assaying creatine kinase 
in terms of preservation in a dissolved state at room temperature. 
Although the glucokinase/glucose-6-phosphate dehydrogenase method somewhat 
resolved the problem of instability of the reagent after dissolution that 
was associated with the aforesaid hexokinase/glucose-6-phosphate 
dehydrogenase method, the stability of the reagent was still insufficient 
and a relatively large quantity of enzymes was required for maintaining 
the stability of the reagent for extended periods of time. In addition, 
with the recent increase of diseases that need urgent assays in clinical 
laboratories, such as myocardial infaraction there has been a pressing 
demand for development of a reagent which enables accurate and rapid 
determination of creatine kinase activity in vital body fluids. In other 
words, high stability in the form of a solution for a long period of time 
has been required of a reagent so that creatine kinase activity can be 
assayed in any time of emergency without requiring adjustment of a reagent 
for minimizing measurement errors. 
SUMMARY OF THE INVENTION 
An object of this invention is to provide a reagent for assaying creatine 
kinase which is excellent in stability during preservation in the form of 
a solution. 
As a result of extensive and intensive investigations for satisfying the 
above-described requirements, the present inventors have found that 
stability of a reagent for assaying creatine kinase after preparation can 
greatly be improved by preparing a first reagent comprising glucokinase, 
glucose-6-phosphate dehydrogenase, NAD(P), ADP and glucose and a second 
reagent comprising creatine phosphate, and maintaining the pH of the 
second reagent within a specific range. 
It has also been found through further stucies that the stability of the 
reagent can further be improved by incorporating glucokinase and glucose 
in the second reagent in place of the first reagent, and thus reached the 
present invention. 
That is, the present invention relates to a reagent system for assaying 
creatine kinase, consisting essentially of a first reagent comprising 
glucose-6-phosphate dehydrogenase, .beta.-nicotinamideadenine dinucleotide 
(phosphate), and adenosine diphosphate, and a second reagent comprising 
creatine phosphate, said second reagent being maintained at a pH of from 
7.5 to 10, and at least one of said first reagent and said second reagent 
containing glucokinase and glucose. The reagent system includes two 
preferred embodiments, i.e., a creatine kinase-assaying reagent system 
consisting essentially of a first reagent comprising glucokinase, 
glucose-6-phosphate dehydrogenase, NAD(P), ADP and glucose, and a second 
reagent comprising creatine phosphate, said second reagent being 
maintained at a pH of from 7.5 to 10, and a creatine kinase-assaying 
reagent system consisting essentially of a first reagent comprising 
glucose-6-phosphate dehydrogenase, NAD(P) and ADP, and a second reagent 
comprising glucokinase, creatine phosphate and glucose, said second 
reagent being maintained at a pH of from 7.5 to 10. 
The creatine kinase-assaying reagent according to the present invention can 
be prepared and preserved in large quantities, and thus can promptly cope 
with urgent assaying requirements, since the reagent system has 
significantly increased stability in a dissolved state. 
DETAILED DESCRIPTION OF THE INVENTION 
Glucokinase which can be used in the present invention is not limited in 
source of supply, and includes glucokinase originated from microorganisms, 
e.g., Aerobacter aerogenes, animals, and the like. In particular, 
glucokinase produced by microorganisms whose optimum growth temperature 
ranges from 50.degree. to 85.degree. C. are preferred. Such microorganisms 
include, for example, the genus Bacillus, e.g., Bacillus 
stearothermophilus, B. thermoproteolyticus, B. acidocaldarius, etc.; the 
genus Thermoactinomyces; the genus Thermus; the genus Thermomicrobium; and 
the like. The preferred among these microorganisms is Bacillus 
stearothermophilus, and specific examples thereof ATCC 7933 (ATCC: The 
American Type Culture Collection, Maryland, U.S.A.), ATCC 7954, ATCC 8005, 
ATCC 10194, ATCC 12980, NCA 1503 (NCA: National Canners' Association, 
Washington, D.C., U.S.A.), UK 563 (FERM P-7275, deposited at Fermentation 
Research Institute, Agency of Industrial Science and Technology, Ibaragi, 
Japan, on Sept. 29, 1983), etc. 
Glucose-6-phosphate dehydrogenase which can be used in the present 
invention is also not limited in source of supply, but it is preferable to 
use glucose-6-phosphate dehydrogenase which acts not only on NADP but also 
on NAD as a coenzyme, such as those originated from Leuconostoc 
mesenteroides, Pseudomonas fluorescens, etc.; and more preferably 
glucose-6-phosphate dehydrogenase originated from a thermophilic bacterium 
which acts on both NAD and NADP and is high in stability and 
preservability (as described, for example, in U.S. patent application Ser. 
No. 205,021, now abandoned (corresponding to Canadian Pat. No. 1,156,570 
and Japanese Patent Application (OPI) No. 68391/81), and U.S. Pat. No. 
4,331,762 (corresponding to Japanese Patent Application (OPI) No. 
151491/81)). 
Glucokinase and glucose-6-phosphate dehydrogenase can be prepared from the 
above-described sources by an appropriate combination of known techniques 
including extraction, purification, and so on, for example, a method of 
producing glucokinase as described in U.S. Pat. No. 4,331,762 and Japanese 
patent application (OPI) No. 91190/82, and a method of producing 
glucose-6-phosphate dehydrogenase as described in U.S. patent application 
Ser. No. 205,021, now abandoned (corresponding to Japanese patent 
application (OPI) No. 68391/81 and Canadian Pat. No. 1,156,570) and U.S. 
Pat. No. 4,331,762 (corresponding to Japanese Patent Application (OPI) No. 
151491/81. 
In the present invention, it is necessary to divide the reagent system that 
causes an enzymatic reaction of creatine kinase and participates in an 
enzymatic reaction leading to the production of NAD(P)H necessary for 
determination of UV absorption into a first reagent and a second reagent. 
In one embodiment according to the present invention, the first reagent 
comprises glucokinase, glucose-6-phosphate dehydrogenase, NAD(P), ADP and 
glucose, and, in general, may further contain additives, such as an 
accelerator, an activator, etc. Such additives are conventional and 
include magnesium salts, e.g., magnesium acetate, magnesium sulfate, etc.; 
thiol compounds, e.g., N-acetylcysteine, glutathione, 
2-aminoethylisothiouronium bromide, thioglycolic acid, cysteine, 
mercaptoethanol, dithiothreitol, dithioerythritol, etc.; sodium azide as 
an antiseptic; and the like. Besides, stabilizers, such as polysaccharides 
and derivatives thereof, e.g., soluble starch, methyl cellulose, 
carboxymethyl cellulose, etc.; proteins, e.g., albumin, .gamma.-globulin, 
etc.; and water-soluble high polymeric compounds, e.g., polyvinyl alcohol, 
polyethylene glycol, etc., can also be used appropriately. The second 
reagent comprises creatine phosphate and may further contain known 
additives, such as sodium azide as an antiseptic. 
According to another embodiment of the present invention, the first reagent 
comprises glucose-6-phosphate dehydrogenase, NAD(P) and ADP, and, in 
general, may further contain additives, such as an accelerator, an 
activator, etc. As the additives, any of those enumerated above for the 
first embodiment can be used. Further, all of the above-described 
stabilizers may also be used. 
The second reagent comprises glucokinase, creatine phosphate and glucose, 
and may generally contain additives, such as an accelerator, an activator, 
etc. The additives that can be used are the same as those recited for the 
first embodiment. The same stabilizers as used in the first embodiment may 
also be employed. 
In either of the first and second embodiments of the present invention, all 
components in the first reagent are dissolved in a buffer solution 
preferably having a pH of from 5.5 to 7.4. The buffer solution which can 
be used is not particularly restricted as long as it has a pH value of 
from 5.5 to 7.4, and includes, for example, imidazole-acetic acid, 
tris-acetic acid, triethanolamine-acotic acid, triethanolamine-NaOH, 
morpholinopropanesulfonic acid, morpholinoethanesulfonic acid, etc. Of 
these, the first four of the noted buffer solutions are more advantageous 
in the first reagent. 
All components in the second reagent should be dissolved in a buffer 
solution of pH 7.5 to 10. The buffer solution which can be used is not 
particularly limited as long as it has a pH of 7.5 to 10, and includes, 
for example, tris-acetic acid, triethanolamine-NaOH, glycine-KOH, vicine, 
etc. Of these, the first two buffer solutions are used to advantage in the 
second reagent. 
The concentration of the buffer solution for each of the first and second 
reagents can be selected so that a mixture of the first reagent and the 
second reagent in selected proportions may have an optimal pH value for 
creatine kinaso to be assayed, i.e., of from 6 to 7.2. The first and 
second reagents are generally mixed in a volumetric ratio range of from 
2/1 to 10/1, and preferably from 2/1 to 8/1. The concentration of the 
buffer solution each of the first and second reagents can be selected 
through simple experiments by fixing a mixing proportion to, e.g., 4/1 by 
volume; a pH of the first reagent to, e.g., 6.7; a pH of the second 
reagent to, e.g., 8.5; and a pH of the final reagent mixture to, e.g., 6.7 
to 6.8. For example, the object can be achieved by using a 150 mM 
imidazole-acetic acid buffer solution (pH 6.7) for a first reagent and a 
25 mM trisacetic acid buffer solution (pH 8.5) for a second reagent.

Specific examples of the first and second reagent formulations according to 
the first and second embodiments of this invention are shown below, but 
the present invention is not to be deemed to be limited thereto. 
FIRST EMBODIMENT 
First Reagent: 
Imidazole-acetic acid buffer solution 
Magnesium acetato 
Ethylenediaminetetraacetic acid (EDTA) 
ADP 
NAD(P) 
Adenosine monophosphate (AMP) 
Glucose 
Adenosine pentaphosphate 
N-Acetylcysteine 
Glucokinase 
Glucose-6-phosphate dehydrogenase 
Sodium azide 
Second Reagent: 
Tris-acetic acid buffer solution 
Creatine phosphate 
Sodium azide 
SECOND EMBODIMENT 
First Reagent: 
Imidazole-acetic acid buffer solution 
Magnesium acetate 
EDTA 
ADP 
NAD(P) 
AMP 
Adenosine pentaphosphate 
N-Acetylcysteine 
Glucose-6-phosphate dehydrogenase 
Sodium azide 
Second Reagent: 
Tris-acetic acid buffer solution 
Magnesium acetate 
EDTA 
Creatine phosphate 
Glucose 
Glucokinase 
Sodium azide 
The concentrations of each component for the creatine kinase-assaying 
reagent of the present invention can be selected according to known 
techniques. In general, from 0.1 to 40 unit/ml, and preferably from 0.2 to 
20 unit/ml, of glucokinase; from 0.1 to 40 unit/ml, and preferably from 
0.2 to 20 unit/ml, of glucose-6-phosphate dehydrogenase; from 2 to 70 mM, 
and preferably from 5 to 40 mM, of creatine phosphate; from 0.1 to 20 mM, 
and preferably from 0.2 to 10 mM, of ADP; from 0.05 to 20 mM, and 
preferably 0.1 to 10 mM, of NAD(P); from 1 to 200 mM, and preferably from 
2 to 100 mM, of glucose; from 0.5 to 30 mM, and preferably from 2 to 15 
mM, of a magnesium salt; from 0.5 to 50 mM, and preferably from 2 to 30 
mM, of a thiol compound; from 0.2 to 20 mM, and preferably from 0.5 to 15 
mM, of AMP; from 1 to 100 .mu.M, and preferably from 2 to 50 .mu.M, of 
adenosine pentaphosphate; from 0.1 to 20 mM, and preferably from 0.2 to 10 
mM, of EDTA; and from 0.5 to 50 mM, and preferably from 1 to 30 mM, of 
sodium azide can be used. 
According to the present invention, stability of the reagent system can be 
significantly improved by dividing the reagent components including 
glucokinase, glucose-6-phosphate dehydrogenase, NAD(P), ADP, glucose and 
creatine phosphate into two reagents and by controlling the pH value of 
the second reagent within a specific range. The thus improved stability 
makes it possible to prepare a large quantity of a reagent system at one 
time, thus providing an ability to cope with urgent clinical examinations. 
Further, the capability of preparing a reagent system in large quantities 
results in improvement of working efficiency and reduction of occurrences 
of discarding surplus reagent. Thus, the creatine kinase-assaying reagent 
in accordance with the present invention provides a very valuable 
contribution to the field of clinical examinations. Furthermore, the 
present invention has an effect on achieving a great saving of resources, 
since the amounts of enzymes and other expensive reagents required for 
assaying can be reduced. 
The present invention will now be illustrated in greater detail with 
reference to Examples and Comparative Examples, but it should be 
understood that the present invention is not limited thereto. 
EXAMPLE 1 AND COMATIVE EXAMPLE 1 
A first reagent was prepared from 1.4 unit/ml of glucokinase produced by 
Bacillus stearothermophilus (manufactured by Seikagaku Kogyo Co., Ltd.), 
1.2 unit/ml of glucose-6-phosphate dehydrogenase produced by Leuconostoc 
mesenteroides (manufactured by Oriental Yeast Industry Co., Ltd.), 1.25 mM 
of ADP disodium salt, 0.75 mM of NADP sodium salt, 25 mM of glucose, 6.25 
mM of AMP, 12.5 .mu.M of adenosine pentaphosphate, 12.5 mM of 
N-acetylcysteine, 12.5 mM of magnesium acetate, 10 mM of sodium, azide, 
2.5 mM of EDTA, and 150 mM of an imidazole-acetic acid buffer solution (pH 
6.7). Then, a second reagent was prepared from 100 mM of creatine 
phosphate, 10 mM of sodium azide, and 25 mM of a tris-acetic acid buffer 
solution (pH 8.5). 
Both the first and second reagents were allowed to stand in a thermostat at 
30.degree. C., and the first reagent and the second reagent were mixed at 
a ratio of 4/1 by volume upon use to prepare a creatine kinase-assaying 
reagent for assaying creatine kinase activity in blood serum (Example 1). 
For comparison, a creatine kinase-assaying reagent of one-reagent type was 
prepared from 3 unit/ml of the same glucokinase as used above, 3 unit/ml 
of the same glucose-6-phosphate dehydrogenase as used above, 1.0 mM of ADP 
disodium salt, 1.6 mM of NADP sodium salt, 20 mM of glucose, 5 mM of AMP, 
10 .mu.M of adenosine pentaphosphate, 10 mM of N-acetylcysteine, 10 mM of 
magnesium acetate, 10 mM of sodium azide, 2 mM of EDTA, 20 mM of creatine 
phosphate and 120 mM of an imidazole-acetic acid buffer solution (pH 6.7). 
The comparative reagent was allowed to stand in a thermostat at 30.degree. 
C., and a requisite amount thereof was taken therefrom to assay creatine 
kinase activity in blood serum (Comparative Example 1). 
A 0.5 ml portion of each of the thus prepared creatine kinase-assaying 
reagents kept at 30.degree. C. was placed in a cell having a light path 
length of 1 cm, and 20 .mu.l of a commercially available standard serum 
was added thereto. The creatine kinase activity of the specimen was 
assayed based on the change of absorbance at 340 nm by means of a 
spectrophotometer kept at 30.degree. C. The creatine kinase activity 
obtained on the day of preparing the reagent (0 day) was taken as 100%, 
and changes in the assayed values with the passage of time were relatively 
traced while maintaining the reagents at 30.degree. C. 
The results obtained revealed that the creatine kinase activity could be 
substantially 100% detected over a period of 18 days from the day of 
preparing the reagent in Example 1, while, in Comparative Example 1, the 
creatine kinase activity could be substantially 100% detected over a 
period of only 10 days from the day of preparing the reagent. 
The "days" used herein refers to a period (days) of the reagents used, 
i.e., a period that creatine kinase activity could be substantially 100% 
detected. Since the values to be obtained thereafter were an unreliable 
value, the reagents used were discarded. 
It is apparent from these results that the stability of the reagent in the 
form of a solution can conspicuously be increased in accordance with the 
present invention by dividing the reagent into a first reagent and a 
second reagent and by controlling the pH of the second reagent. It can 
also be seen that reduction in requisite amounts of expensive reagents, 
such as glucokinase, glucose-6-phosphate dehydrogenase, NADP, etc., can be 
realized by the present invention. 
COMATIVE EXAMPLE 2 
A first reagent was prepared in the same manner as in Example 1. A second 
reagent was prepared from 100 mM of creatine phosphate, 10 mM of sodium 
azide and 25 mM of a tris-acetic acid buffer solution (pH 7.0). 
Both the reagents were allowed to stand in a thermostat at 30.degree. C., 
and the first reagent and the second reagent were mixed at a proportion of 
4/1 by volume when in use to assay creatine kinase activity in blood serum 
in the same manner as in Example 1. 
As a result, it was found that the creatine kinase activity could be 
substantially 100% detected over a period of only 12 days. 
It can be seen from the above results that remarkable improvement in 
stability of the reagent after dissolution can be established as in 
Example 1 not only by dividing a creatine kinase-assaying components into 
two reagents but also controlling the pH of the second reagent within a 
specific range. 
EXAMPLE 2 
A first reagent was prepared from 1.2 unit/ml of glucose-6-phosphate 
dehydrogenase produced by Leuconostoc mesenteroides (manufactured by 
Oriental Yeast Industry Co., Ltd.), 1.25 mM of ADP disodium salt, 0.75 mM 
of NADP sodium salt, 6.25 mM of AMP, 12.5 .mu.M of adenosine 
pentaphosphate, 12.5 mM of N-acetylcysteine, 10 mM of magnesium acetate, 
10 mM of sodium azide, 2 mM of EDTA and 150 mM of an imidazole-acetic acid 
buffer solution (pH 6.7). Then, a second reagent was prepared from 100 mM 
of creatine phosphate, 10 mM of sodium azide, 10 mM of magnesium acetate, 
2 mM of EDTA, 100 mM of glucose, 5.6 unit/ml of commercially available 
glucokinase (manufactured by Seikagaku Kogyo Co., Ltd.) and 25 mM of a 
tris-acetic acid buffer solution (pH 8.5). 
Both the reagents were allowed to stand in a thermostat at 30.degree. C., 
and were used to assay creatine kinase activity in blood serum in the same 
manner as described in Example 1. 
As a result, 100% of the creatine kinase activity could be substantially 
detected over a period of 20 days from the day of preparing the reagent, 
indicating that incorporation of glucokinase and glucose in the second 
reagent further improves stability of the reagent after dissolution. 
EXAMPLES 3 AND 4 
Relative changes of creatine kinase activity with the passage of time were 
traced in the same manner as described in Example 1 (Example 3) or Example 
2 (Example 4) except that the reagents were preserved at 4.degree. C. 
As a result, it was revealed that the creatine kinase activity could be 
substantially 100% detected over a period of about 60 days in Example 3 
and about 70 days in Example 4. 
EXAMPLE 5 
The same procedures as in Example 2 were repeated except that the second 
reagent contained 150 mM of creatine phosphate. 
As a result, the creatine kinase activity could be substantially 100% 
detected over a period of 24 days from the day of preparing the reagents. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.