JPH11253193A - Test piece for assaying creatine kinase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject test piece capable of assaying creatine kinase activity in high sensitivity over a wide assaying range by including a dehydrogenase, diaphorase, NAD(P), and a water-soluble tetrazolium in a filmy base. SOLUTION: This test piece for assaying creatine kinase activity is obtained by the following procedure: a dehydrogenase, diaphorase, NAD or NADP, a water-soluble tetrazolium such as 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, creatine phosphate and others are acimixed in 100 mM of an imidazoleacetate buffer solution (pH 6.7), and the resulting solution is dripped onto a filmy base such as polyethylene terephthalate film followed by air-drying; subsequently, the resulting filmy base such as palyetylene terephthalate film is coated with a 1 vol.% methanol solution of vinylpyrrolidone-vinyl acetate copolymer followed by a methanol suspension of cellulose powder, and the filmy base thus coated is dried at 40-50 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a test strip for measuring creatine kinase activity in a sample.

[0002]

2. Description of the Related Art In recent years, reagents utilizing an enzymatic reaction have been widely used as reagents for examining and diagnosing disease states, instead of conventionally used organic chemical reagents.
A reagent utilizing an enzyme reaction utilizes the property of an enzyme to specifically convert a specific component in a living body into a detectable substance, and is usually a substance to be measured (A)
Is converted to an intermediate product (I) using an enzyme (a) specific thereto.
-1). The intermediate (I-1) is further reacted with a specific enzyme (i-1) to react with the intermediate (I-1).
2) The reaction of conversion to 2) is repeated and finally converted to a detectable substance (F), and this detectable substance (F) is quantified using a spectrophotometer or the like, or from a change in color tone due to the naked eye or the like. It is.

[0003]

Embedded image

In such reagents, the detectable substance (F) includes NAD and its reduced form (NADH), and their analogous compounds NADP and its reduced form (NAD).
DPH) and the like are widely used (hereinafter, these compounds are collectively referred to as nicotine nucleotides), and these nicotine nucleotides are ultraviolet (34)
Since a change in absorbance is observed at around 0 nm), a reagent for measuring the generated or reduced nicotine nucleotides using a spectrometer has been proposed. Also, the generated NA
Many reagents have been reported which convert tetrazolium to formazan by measuring DH or NADPH with diaphorase to measure in the visible region [for example, bile acids (JP-A-60-214900, Clinical Chemistry No. 19).
Volume, pp. 290-299 (1990)), triglycerides (JP-A-55-14899), alcohols (JP-B-4-3947), and amylase (JP-B-63-3).
No. 7640), creatine kinase (Japanese Unexamined Patent Application Publication No.
No. 16699), NAD (P) H (Japanese Patent Publication No. 4-70)
000), polyamines (Japanese Patent Publication No. 6-68490), glucose (Japanese Patent Publication No. 7-34757),
Benzylamine (JP-A-7-184693)].
Also, a test piece for measuring creatine kinase activity has been reported (Japanese Patent Application Laid-Open No. 63-283600,
JP-B-6-95959, JP-A-1-320999
No.).

However, these reagents include dichlorophenol indophenol (hereinafter referred to as DCI) as a coloring reagent.
Abbreviated as P. ), Tetrazolium blue, neotetrazolium blue, MTT, INT, and nitrotetrazolium blue. Among the above color forming reagents, DCIP changes from a colored state due to the action of diaphorase to a colorless state, and thus is not suitable for preparing test pieces. In addition, tetrazolium, which is used as another color-forming reagent, has low solubility in water, so it is not easy to prepare a test piece (see Comparative Example). Therefore, an operation such as diluting the sample to be measured was required (for example, Medical Technology Magazine, Vol. 11, No. 6, 496-50).
On page 5, it is described that when measuring creatine kinase activity, the activity can be measured only up to 1000 units / l even if the sample is diluted 9-fold. ). In these test strips, since the blood, urine, and the like, which are the test specimens, are aqueous, even if the test specimens are directly added to the test specimens and the reflected light is measured, the activity is measured with high sensitivity and reproducibility. There was a problem that it was extremely difficult.

On the other hand, in examples other than enzymes such as creatine kinase, there is an example in which a reducing color former is used for a test piece for measuring magnesium, which is an inorganic substance, and a tetrazolium salt is mentioned as an example of the reducing color former. There are some (for example, JP-A-9-266796, JP-A-9-266).
797), only tetrazolium violet is a highly water-soluble tetrazolium salt among the exemplified tetrazolium salts, and all others have low solubility in water. In addition, when other representative water-soluble tetrazolium is used as a coloring agent in this test piece, spontaneous coloring occurs, so it is inappropriate to use water-soluble tetrazolium as a coloring reagent in the test piece. It was considered.

[0007] It is an object of the present invention to provide a test strip capable of measuring creatine kinase activity with high sensitivity over a wide measurement area by measuring reflected light.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a test piece for measuring creatine kinase activity using a water-soluble tetrazolium which is considered to be inappropriate. Was further found to be able to measure creatine kinase activity with high sensitivity over a wide measurement range by using water-soluble tetrazolium, and reached the present invention. That is, the first invention includes at least dehydrogenase, diaphorase, NAD or NA.
A gist of a test strip for measuring creatine kinase activity, comprising a DP, a water-soluble tetrazolium, and a carrier. In the second invention, the water-soluble tetrazolium is 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2.
H-tetrazolium, 2- (4-nitro, 2-methoxyphenyl) -3- (4-nitrophenyl) -5- (2,
4-disulfophenyl) -2H-tetrazolium, 2,
3,5-triphenyl-2H-tetrazolium or 2,
The gist of the test piece is 5-diphenyl-3- (1-naphthyl) -2H-tetrazolium.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The dehydrogenase used in the present invention is not particularly limited as long as it is a dehydrogenase that specifically acts on glucose 6-phosphate, and examples include glucose 6-phosphate dehydrogenase. There are no particular limitations on the species of the organism as the source, and examples thereof include those derived from microorganisms such as lactic acid bacteria and Zymomonas.

[0010] The water-soluble tetrazolium used in the present invention is not particularly limited as long as it has a solubility in water of 5 mM or more and is colored by being reduced by the action of diaphorase in the presence of NADH or NADPH. Instead, for example, 2- (4-iodophenyl)
-3- (4-Nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, 2- (4-nitro, 2-methoxyphenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, 2,3,5-triphenyl-2H-tetrazolium or 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium, 2- (4- Iodophenyl) -3- (2,4-dinitrophenyl) -5- (2
4-disulfophenyl-2H-tetrazolium, 2-
(2-methoxy, 4-carboxyphenyl) -3-benzothiazyl-5- (4-sulfoethylaminocarbonylphenyl) -2H-tetrazolium, 3,3′-
[3,3'-Dimethoxy- (1,1'-biphenyl)-
4,4′-diyl] -bis [2-benzothiazyl-4-
(4-disulfoethylaminocarbonylphenyl)-
Tetrazolium]. These water-soluble tetrazoliums are WST-1, WST-8, tetrazolium red (hereinafter abbreviated as TR), tetrazolium violet (hereinafter abbreviated as TV), and WST, respectively.
-3, WST-4, and WST-5, and are commercially available or supplied as samples from Dojindo Chemical Laboratories or Sigma-Aldrich. Among these water-soluble tetrazoliums, WST-1, WST-8, TR, and TV are preferable because they can be uniformly fixed to a test piece. Also,
WST-1, WST-8, and TR are particularly preferable because they can be easily dissolved in a solution or a solvent and can be retained on a test piece in a sufficient amount. Below, WST-1, WST-
8, the structural formulas of TR and TV are shown.

[0011]

Embedded image

[0012]

Embedded image

[0013]

Embedded image

[0014]

Embedded image

The diaphorase used in the present invention is not particularly limited as long as it catalyzes the above-mentioned reaction, and the source species thereof is not particularly limited.
Examples thereof include those derived from microorganisms such as Bacillus stearothermophilus and Clostridium kluyberg and those derived from pig heart. Among them, diaphorase derived from a thermophilic microorganism is preferable because of its excellent storage stability, and specifically, diaphorase derived from Bacillus stearothermophilus is preferably used.

The diaphorase used in the present invention includes formazan and oxidized nicotine nucleotide from tetrazolium and reduced nicotine nucleotide (NADH or NADPH) calculated by the following formula in order to improve the accuracy of creatine kinase activity measurement. (NAD or N
It is preferable to use one having a reaction equilibrium constant (K value) of 1 or more, preferably 10 or more, more preferably 100 or more in the direction toward ADP). For example, diaphorase I or diaphorase derived from Bacillus stearothermophilus
Preferably, II is used.

[0017]

Embedded image

[0018] These components have an enzymatic reaction to formazan of 70% or more from a reaction involving creatine kinase,
The diaphorase is preferably blended so as to progress preferably at least 90%, more preferably at least 95%, and as diaphorase, it is preferably 0.1 to 1,000,000 units / l, more preferably 0.1 to 10,000 units / l. Is 1 to 10
An enzyme solution having a concentration of 00 units / l was applied to a test piece 100 cm ²
0.1 to 10,000 μl, preferably 1 to 1000 μl,
More preferably, it may be contained in an amount of 1 to 100 μl. The dehydrogenase may be at the same level as the concentration of diaphorase. As NAD or NADP, 0.0
01 nM to 200 mM, more preferably 0.1 nM to
A 50 mM solution was applied at 0.1 to 1 per 100 cm ² of the test piece.
It may be contained in an amount of 10,000 μl, preferably 1 to 1000 μl, more preferably 1 to 100 μl.

The amount of the water-soluble tetrazolium is 0.01 to 500 mg, preferably 0.1 to 100 mg, more preferably 0.1 to 50 mg per 100 cm ² of the test piece.
g may be contained. At this time, if the amount of the water-soluble tetrazolium is too small, the coloring becomes low, and if it is excessive, it becomes insoluble and the accuracy may be lowered, which is not preferable.

Specifically, when measuring creatine kinase activity, enzymes and substrates may be contained so that creatine kinase activity can be measured, for example, according to the following reaction formula (1).

[0021]

Embedded image

Since the test strip of the present invention contains at least dehydrogenase and diaphorase as enzymes and NAD or NADP as substrates and water-soluble tetrazolium, other enzymes such as hexokinase or glucokinase, Creatine phosphate which is a substrate,
Glucose and ADP may be included as needed.
In addition, ascorbic acid oxidase, N-acetylcysteine, magnesium ion and the like may be coexisted as necessary other than the above enzymes and substrates.

When the test strip of the present invention contains the above enzymes, substrates, etc., the enzymes necessary for the carrier constituting the test strip,
What is necessary is just to impregnate all the substrates and the like. When impregnating, the same part of the carrier constituting the test piece may be impregnated with all necessary enzymes, substrates, etc., and a part of the necessary enzymes, substrates, etc. A part of the carrier constituting the test piece may be impregnated, and the remaining necessary enzymes, substrates and the like may be separately impregnated into the other part of the carrier constituting the test piece.

Of the above enzymes, substrates and the like, it is sometimes preferable to impregnate N-acetylcysteine and water-soluble tetrazolium into different parts of the carrier constituting the test piece from the viewpoint of stability during storage. . When glucose-6-phosphate dehydrogenase is used as the dehydrogenase, it may be preferable to impregnate glucose and glucose-6-phosphate dehydrogenase in different parts of the carrier for the same reason. Furthermore, it may be preferable to impregnate magnesium ion and dehydrogenase, NAD and creatine phosphate in different parts of the carrier for the same reason.

In this case, the concentration of each enzyme coexisting on the test piece may be in the same range as the concentration of diaphorase. The concentration of a substrate such as NAD or NADP or ADP is 0.1 to 100 mM, preferably 0.1 to 20 mM.
M range from 0.1 to 1 per 100 cm ^{2 of} test specimen.
It may be contained in an amount of 10,000 μl, preferably 1 to 1000 μl, more preferably 1 to 100 μl.

In the present invention, the test piece may contain a buffer, an activator, a stabilizer, a thickener and other additives for adjusting the pH. Examples of the buffer include potassium phosphate, imidazole and the like.
-A good buffer such as morpholineethanesulfonic acid (MES), N, N-bis- (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES).

As the activator, for example, Triton X-1
Nonionic, anionic or cationic surfactants such as 00 and Twin 20 are used. Such an activator contributes to the storage stability and the blank value of the test piece. The concentration of these activators is 0.001-2.
0%, preferably 0.01-5% solution,
0.1-10000 μl per 0 cm ² , preferably 1-100
0 μl, more preferably 1 to 100 μl, may be contained.

Examples of the stabilizer include proteins such as bovine serum albumin, maltose, glucose, and the like.
Saccharides such as sucrose, high molecular compounds such as polyethylene glycol, and metal ions such as magnesium, potassium, and calcium are used. Metal ions also act as activators for enzymes. Further, ethylenediaminetetraacetic acid (E
DTA), ethylene glycol (β-aminoethyl ether) tetraacetic acid (EGTA) and the like can also be used. As to the concentration of these stabilizers, sugar is in the range of 0.1 to 50% by weight, preferably 1 to 25% by weight, and protein is in the range of 0.001 to 50% by weight, preferably 0.1 to 25% by weight. range, metal ions 0.001～10MM, preferably in the range of 0.1-10 mM, EDTA and EGTA are 0.001～10MM, preferably a solution in the range of 0.1 to 2 mm, the test piece 100 cm ² per 0 .1 to 10,000 μ
1, preferably 1 to 1000 μl, more preferably 1
１００100 μl may be contained.

When the test piece of the present invention contains additives such as the above-mentioned activator and stabilizer, the additives together with necessary enzymes and substrates are impregnated into a carrier constituting the test piece. I just need. When impregnating, all necessary enzymes, substrates, additives, etc. may be impregnated into the same part of the carrier constituting the test piece, and among the necessary enzymes, substrates, additives, etc. May be impregnated into a part of the carrier constituting the test piece, and the remaining necessary enzymes, substrates, additives and the like may be separately impregnated into the other part of the carrier constituting the test piece.

When EDTA is added to the test piece of the present invention, if creatine phosphate or N-acetylcysteine is additionally contained, EDTA and creatine phosphate or N-acetylcysteine are added from the viewpoint of storage stability. In some cases, it is preferable to impregnate acetylcysteine into different portions of the carrier constituting the test piece.

As the carrier used for the test piece of the present invention, a known carrier composed of a polymer material can be used, and specific examples include papermaking and nonwoven fabrics composed of natural fibers and synthetic fibers, and membrane filters. Can be Examples of the membrane filter include a nitrocellulose-based membrane having a pore size of about 0.1 to 0.5 μm, a cellulose acetate-based membrane, a polyvinyl alcohol-based membrane, and the like.

The test piece of the present invention may be prepared, for example, as follows. That is, it can be produced by dissolving the above components in water, a buffer solution or the like, impregnating the carrier, and then sufficiently removing the water content of the carrier by freeze-drying or the like.

The test piece prepared in this manner can be used after being cut into small pieces, or it can be pasted on another carrier and used as a cassette. Furthermore, it is also possible to work on a pad.

The subject of the test strip of the present invention is not particularly limited. When measuring creatine kinase activity in a biological component, a biological sample such as blood, plasma, serum, or urine may be used as a specimen.

Measurement of creatine kinase activity in a biological component using the test strip of the present invention can be performed, for example, as follows. That is, (1) a fixed amount of the specimen containing the substance to be measured is dropped on the test piece. (2) After a certain period of time has elapsed from the dropping of the sample, it is confirmed that the sample has sufficiently permeated the test piece, and light of a certain wavelength is irradiated using an appropriate reflected light measuring device to measure the reflection intensity.
(3) A standard curve is prepared in the same manner by measuring a standard amount of the measurement target substance, and the concentration of the measurement target substance in the sample is calculated from the calibration curve.

[0036]

Next, the present invention will be described specifically with reference to examples. Diaphorase I from Bacillus stearothermophilus used in the present invention (Product No. 100436),
II (product number 100377) and glucokinase (product number 120387) were purchased from Seikagaku Corporation. Diaphorase derived from Clostridium kluyberg (Product No. D5540) and diaphorase derived from pig heart (Product No. D3752) were purchased from Sigma.

Hexokinase (product number 142636)
2), glucose 6-phosphate dehydrogenase (hereinafter, referred to as
G6PDH, product number 737208), creatine kinase (product number 126969) is available from Boehringer
Purchased from Mannheim. For each enzyme activity, the value described in the package insert or label at the time of purchase was used as it was.

Tetrazolium blue (hereinafter abbreviated as TB, product number 1871-22-3), neotetrazolium blue (hereinafter abbreviated as NeoTB, product number 298-)
95-3), MTT (product number 2348-71-2),
INT (product number 146-68-9), nitrotetrazolium blue (hereinafter abbreviated as NTB, product number 298-)
83-9), WST-1 (product number 150849-52)
-8) was purchased from Dojindo Laboratories. WST-8 was obtained as a sample from Dojindo Laboratories. Nitro blue tetrazolium (hereinafter abbreviated as NBT, product number N
6876), TV (product number T0174), and TR (product number T84859) were purchased from Sigma-Aldrich. Cellulose powder (product number 075-41) was purchased from Nacalai Tesque. Other reagents used were commercially available special grades.

Reference Example 1 In a 500 mM imidazole buffer (pH 6.7), a final concentration of 50 mM WST-8 and diaphorase 10 derived from Clostridium kluyberg were used.
Unit / ml was prepared, and the solution was dropped on a 5 mm-wide polyurethane film at intervals of about 1 cm (about 20 μl) using a peristaltic pump. The film was dried by passing it through a hot air drying zone at about 50 ° C. (passing time: about 2 minutes), and then cut into a size of 5 × 5 mm around the point of dropping to obtain a test piece. The test piece was bonded at both ends to a form substrate having a 3 × 3 mm window.

2 μl of an aqueous solution containing a predetermined concentration of NADH was dropped on the obtained test piece, and after 5 minutes, the reflectance at 550 nm was measured using a colorimeter (CR-200, manufactured by Minolta). The result is shown in FIG. FIG. 1 is a diagram showing the relationship between NADH concentration and reflectance. From this figure, it can be seen that NADH can be accurately quantified from the reflectance by using the above test piece. From the results, it can be seen that any test substance can be quantified by preparing a test piece in which various dehydrogenases coexist as described in the specification.

Example 1 A test piece for creatine kinase quantification was prepared as follows. That is, a final concentration of 30 mM creatine phosphate, 50 mM glucose, 5 mM NAD, 5 mM in 100 mM imidazole acetate buffer (pH 6.7).
M ADP, 10 mM magnesium acetate, 2 mM E
DTA-2Na, 25 mM NAC, 4 wt% WST
-1 was dissolved. About 30000 in about 350 μl of this solution
2 μl of diaphorase solution from U / ml porcine heart and 1
10 μl of G6PDH at 650 units / ml, 3600
1 μl of unit / ml hexokinase was added. This solution was dropped on a polyethylene terephthalate film using a pipetteman, air-dried, and then 1% by volume of methanol of vinylpyrrolidone vinyl acetate copolymer (20:80 in weight ratio) was applied.
It was suspended in methanol and applied in a small amount. This is 40-50
After drying at ℃, a test piece of a polyethylene terephthalate support membrane was obtained. A creatine kinase solution was added to the test piece for 5 minutes.
μl was dropped, and the reflectance was measured every 30 seconds.

A calibration curve showing the relationship between NADH concentration and reflectance was prepared in the same manner as in Reference Example 1 except that WST-1 was used in place of WST-8. The amount of change corresponding to NADH per unit time was calculated. The result is shown in FIG. FIG. 2 is a diagram showing the results of creatine kinase activity measurement, in which the vertical axis indicates the amount of change corresponding to NADH, and the horizontal axis indicates creatine kinase activity. From this figure, it can be seen that quantification of creatine kinase of at least 3000 units / l is possible by using the test piece of the present invention.

Example 2 A test piece for creatine kinase quantification was prepared as follows. That is, a final concentration of 30 mM creatine phosphate, 50 mM glucose, 20 mM NAD, 100 mM imidazole acetate buffer (pH 6.7) was added to 100 mM imidazole acetate buffer (pH 6.7).
0 mM ADP, 10 mM magnesium acetate, 2 mM
EDTA-2Na, 25 mM NAC, 5 wt% T
R was dissolved. To about 350 μl of this solution, 2 μl of a bacterium stearothermophilus-derived diaphorase I solution of about 30,000 units / ml and 1650 units / ml
Of G6PDH, 2 μl of glucokinase at 2500 units / ml. 2 μl of this solution was dropped on a 3 × 3 mm small piece of polyflon filter paper (PF050) manufactured by Advantech Co. using a pipetman, and freeze-dried to obtain a test piece. The test piece was stuck to the backing with a double-sided tape. 5 μl of a creatine kinase solution was added to the test piece.
1 drop, and the reflectance was measured every 15 seconds.

A calibration curve showing the relationship between NADH concentration and reflectance was prepared in the same manner as in Reference Example 1 except that TR was used in place of WST-8. The amount of change corresponding to NADH per hit was calculated. The test piece (before the creatine kinase diluent was dropped) was wrapped with aluminum foil and allowed to stand in a 37 ° C. dry oven for one month. Similarly, the creatine kinase concentration and the unit time were measured. The relationship of the amount of change corresponding to NADH per unit was examined. The result is shown in FIG. FIG. 3 is a diagram showing the results of measuring the activity of creatine kinase using test pieces immediately after preparation and after standing for one month,
3 in FIG. 3 shows the result when creatine kinase was dropped immediately after the preparation, and ○ shows the result when measured after standing for one month. FIG. 3 shows that the use of the test strip of the present invention enables quantification of creatine kinase of at least 3000 units / l. Further, it can be seen that the test piece of the present invention has excellent storage stability.

Reference Examples 2 to 5 and Comparative Examples 1 to 6 A 100 mM imidazole buffer (pH 6.7) was added to a final concentration of 30 mM each of the tetrazolium shown in Table 1 and diaphorase II1 derived from Bacillus stearothermophilus.
A solution containing 0 units / ml was prepared and was
Then, 3 μl of the solution was dropped onto a Whatman filter paper (No. 2) and dried to prepare a test piece. 5 m
8 mm diameter paper disk immersed in NADH solution of M (product number 49005010, manufactured by Advantech)
Was quickly and horizontally contacted, and the color development of the test piece was visually observed after about 1 minute. Table 1 shows the results.

[0046]

[Table 1]

As is clear from Table 1, when WST-1, WST-8, TR, and TV, which are water-soluble tetrazolium, were used, the dye was uniformly fixed on the test piece. In addition, in terms of ease of preparation of test pieces,
The test piece of the reference example was easier than the test piece of the comparative example.

Example 3 A test piece for creatine kinase quantification was prepared as follows. That is, a final concentration of 25 mM creatine phosphate, 25 mM glucose, 2 mM NADP, 150 mM imidazole acetate buffer (pH 6.6)
mM ADP, 5 mM magnesium acetate, 2 mM E
DTA-2K, 30 mM NAC, 12% by weight of Twin 20, and 1% by weight of WST-1 were dissolved. About 3 of this solution
Bacillus stearothermophilus-derived diaphorase I solution, G6PDH, and glucokinase were added to 50 μl so that the final concentrations would be about 500 units / ml, 10 units / ml, and 10 units / ml, respectively.
2 μl of this solution was dropped on a cellulose acetate membrane (product number A045A090C) manufactured by Advantech Co., Ltd. having a diameter of 6 mm using a pipetman, and freeze-dried to prepare a test piece. An 8 mm diameter paper disk (Advantech, product number 4900501) immersed in a creatine kinase solution of about 3000 units / l was placed on the test piece.
0) was quickly and horizontally contacted, and the reflectance was measured over time as described below, and the creatine kinase activity value was measured.

A calibration curve showing the relationship between NADH concentration and reflectance was prepared in the same manner as in Reference Example 1 except that WST-1 was used in place of WST-8. The relationship between the changes corresponding to NADH per unit time was examined, and the creatine kinase activity value was calculated. The measurement was performed four times, and the reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 3176 ± 0 units / l.

Example 4 Example 3 except that WST-8 was used instead of WST-1.
The creatine kinase activity value was calculated in the same manner as described above, and its reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 2948 ± 61 units / l.

Example 5 The activity of creatine kinase was calculated in the same manner as in Example 3 except that TR was used in place of WST-1, and the reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 2906 ± 109 units / l.

Example 6 The activity of creatine kinase was calculated in the same manner as in Example 3 except that TV was used in place of WST-1, and its reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 2948 ± 61 units / l.

Comparative Example 7 The activity of creatine kinase was calculated in the same manner as in Example 3 except that TB was used in place of WST-1, and its reproducibility was confirmed. As a result, a precipitate was generated in the reagent, and a test piece could not be prepared.

Comparative Example 8 Example 3 except that NeoTB was used instead of WST-1.
The creatine kinase activity value was calculated in the same manner as described above, and its reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 1623 ± 524 units / l, and the variation in the measured value was large. Further, when the state of color development of the test piece was visually observed, it was uneven.

Comparative Example 9 The activity of creatine kinase was calculated in the same manner as in Example 3 except that INT was used in place of WST-1, and its reproducibility was confirmed. As a result of the measurement, the activity value of creatine kinase was 1514 ± 46 units / l, and the dispersion of the measured values was large. In addition, when the state of color development of the test piece was visually observed, the coloring was extremely thin and uneven.

Comparative Example 10 The activity of creatine kinase was calculated in the same manner as in Example 3 except that NTB was used in place of WST-1, and its reproducibility was confirmed. As a result, a precipitate was generated in the reagent, and a test piece could not be prepared.

Comparative Example 11 The activity of creatine kinase was calculated in the same manner as in Example 3 except that NBT was used in place of WST-1, and its reproducibility was confirmed. As a result, a precipitate was generated in the reagent, and a test piece could not be prepared.

Comparative Example 12 The activity of creatine kinase was calculated in the same manner as in Example 3 except that MTT was used in place of WST-1, and its reproducibility was confirmed. As a result, it was not possible to measure a measurement value with a very large variation. Further, when the state of color development of the test piece was visually observed, it was uneven.

Examples 3 to 6, Comparative Examples 7 to 1
From the results of 2, it was possible to measure the activity of creatine kinase in the test piece of the present invention using the water-soluble tetrazolium, whereas in the case of using another tetrazolium, a precipitate was formed in the reagent and the test piece was used. Was not able to be prepared, or even if a test piece could be prepared, the reaction was inhibited or the coloration was uneven and the activity of creatine kinase could not be measured. Further, the test piece of the present invention was easier than the test piece of the comparative example in terms of easiness of preparation of the test piece.

[0060]

The test piece of the present invention can be measured in a wide range of measurement. Further, the test piece of the present invention is excellent in storage stability.

[Brief description of the drawings]

FIG. 1 shows the NADH when the concentration of NADH was measured using a test piece to which WST-8 was added as tetrazolium.
It is a figure which shows the relationship between a density and a reflectance.

FIG. 2 is a graph showing the results when the activity of creatine kinase was measured using the test strip of the present invention.

FIG. 3 is a view showing the results when the activity of creatine kinase was measured using test pieces immediately after preparation and after standing for one month.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hitoshi Kondo 23 Uji Kozakura, Uji-city, Kyoto Prefecture Unitika's Central Research Laboratory

Claims (2)

[Claims] 1. A test strip for measuring creatine kinase activity, comprising at least dehydrogenase, diaphorase, NAD or NADP, and water-soluble tetrazolium. 2. The method according to claim 1, wherein the water-soluble tetrazolium is 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-.
(2,4-disulfophenyl) -2H-tetrazolium, 2- (4-nitro, 2-methoxyphenyl) -3-
(4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, 2,3,5-triphenyl-2H-tetrazolium or 2,5-diphenyl-
The test piece according to claim 1, which is 3- (1-naphthyl) -2H-tetrazolium.