JPH11197A - Dry analytical element for quantitatively determining creatine kinase or its mb isozyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a dry analytical element for quantitatively determining creatine kinase or creatine kinase MB isozyme, small in decrease of sensitivity even in a normal temperature preservation, and thereby capable of being preserved at normal temperature. SOLUTION: This dry analytical element is a dry analytical element for quantitatively determining creatine kinase, having a detecting reagent layer containing creatine phosphate capable of forming adenosine triphosphate(ATP) in the presence of the creatine kinase, adenosine diphosphate(ADP), a buffer compound for providing a pH environment for producing the ATP, and further an indicator composition for forming a compound detectable by a spectroscopic measuring method by reacting the indicator with the formed ATP, and laminated on a supporter, and the creatine phosphate is arranged so as to be separated from the ADP and other detecting reagent components. A creatine kinase MB isozyme can be determined by combining an antibody specifically inhibiting M subunit activities of the creatine kinase therewith.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a dry analytical element for quantifying creatine kinase or for quantifying MB isozyme of creatine kinase.

[0002]

2. Description of the Related Art Diagnosis of progressive muscular atrophy, dermatomyositis, myocardial infarction, etc. is determined by quantifying creatine kinase (CK, also called creatine phosphokinase (CPK)) contained in blood collected from a subject. It has been known for some time that it is possible and has been used in clinical tests. In addition, creatine kinase (CK) includes creatine kinase MM (CKMM) and creatine kinase MB.
(CKMB) and creatine kinase BB (CKB
It is also known that there are three isozymes, B), and that CKMM is mainly present in skeletal muscle, CKMB is mainly present in myocardium, and CKBB is mainly present in brain and spinal cord. Furthermore, CKMB is excreted from the myocardium into the blood when myocardial infarction occurs, and as a result,
It has also been found that the amount of CKMB in blood increases. Therefore, quantification of CKMB in the blood of a subject is a powerful means for examining the occurrence of myocardial infarction.

[0003] As described above, in clinical tests, creatine kinase is quantified (ie, total creatine kinase is quantified) and / or creatine kinase MB isozyme is quantified to provide progressive muscle atrophy for the subject. Since a powerful clue can be obtained for the diagnosis of dermatitis, dermatomyositis, myocardial infarction, etc., studies on such quantification methods have been conducted, and a highly accurate quantification method has been established at present.

[0004] That is, as a method of measuring creatine kinase, serum or plasma is obtained from blood collected from a subject, and the enzyme activity of creatine kinase contained in the serum or plasma is used to detect creatine kinase by a spectroscopic method. This is a method in which a chemical species that can be produced is quantitatively generated, and the amount of creatine kinase in serum or plasma is quantified from the amount of the generated chemical species. More specifically,
The detection reaction system used in this quantification method can be divided into the following two.

1) Detection reaction system 1 Creatine phosphate (CP) and adenosine diphosphate (AD)
P) and creatine present in a test solution (such as serum or plasma) while adjusting the pH environment of the enzymatic reaction using a buffering compound having a buffering capacity in the pH range of 5.5 to 8.5. By contacting with a kinase (CK) to produce an amount of adenosine triphosphate (ATP) in proportion to the amount of creatine kinase (CK).
TP and glucose (Glu) are converted to hexokinase (H
K) to react with glucose-6-phosphate (G
6P) and then reacting this G6P with nicotinamide adenine dinucleotide oxidized form (NAD (P)) in the presence of glucose-6-phosphate dehydrogenase (G6PDH) to reduce nicotinamide adenine dinucleotide (NAD (P) H) is generated, and finally, the amount of the NAD (P) H generated is measured by a spectroscopic measurement method, and creatine kinase (CK) is quantified using a separately prepared calibration curve. Is the way.

Since the accuracy of the spectrophotometric determination of the produced NAD (P) H tends to be insufficient, the NAD (P) H
A method for quantifying creatine kinase in a test solution by further reacting (P) H with a tetrazolium salt to form a formazan dye and quantifying the amount of the formazan dye that has been developed (particularly). Kaisho 63-32
No. 499). The detection reaction system 1 is shown below by a reaction formula.

[0007] CP + ADP → ATP + creatine (in the presence of CK) ATP + Glu → G6P + ADP (in the presence of HK) G6P + NAD (P) → NAD (P) H + 6-phosphogluconic acid (in the presence of G6PDH) ) NAD (P) H + tetrazolium salt → formazan dye + NAD (P)

2) Detection reaction system 2 Creatine phosphate (CP) and adenosine diphosphate (AD)
P) and creatine kinase present in a test solution (such as serum or plasma) while adjusting the pH environment of the enzyme reaction with a buffering compound exhibiting a buffering ability in the pH range of 5.5 to 8.5. CK) to produce an amount of adenosine triphosphate (ATP) that is proportional to the amount of creatine kinase (CK).
Glycerol kinase (HK) with TP and glycerol
To produce L-α-glycerophosphate, and then react this L-α-glycerophosphate with oxygen in the presence of L-α-glycerophosphate oxidase to generate hydrogen peroxide, Finally, the hydrogen peroxide is reacted with the leuco dye to form a blue dye, the amount of the blue dye is measured by a spectroscopic measurement method, and creatine kinase (CK) is prepared using a separately prepared calibration curve. Is a method of quantifying The detection reaction system 2 is shown below by a reaction formula.

CP + ADP → ATP + creatine (in the presence of CK) ATP + glycerol → L-α-glycerophosphate + ADP (in the presence of glycerol kinase) L-α-glycerophosphate + O ₂ → H ₂ O ₂ + dihydroxyaceto Phosphoric acid (in the presence of L-α-glycerophosphate oxidase) H ₂ O ₂ + leuco dye → blue dye + H ₂ O

On the other hand, as described above, since creatine kinase has three types of isozymes, quantification of creatine kinase MB isozyme is not easy. That is, in principle, it is necessary to separate and quantify the MB isozyme from the MM isozyme and the BB isozyme. However, since the BB isozyme is originally present in the brain and spinal cord, its abundance in blood is very small and its presence can be ignored. Therefore, M
Quantification of the B isozyme is possible if the MM isozyme can be separated, but the separation is very difficult. Therefore, instead of separating the MM isozyme in a blood sample containing creatine kinase (such as serum or plasma), a method of inhibiting the activity of the MM isozyme without affecting the activity of the MB isozyme,
A method for realizing substantial MB isozyme separation has already been developed (see Japanese Patent Publication No. 56-19239). In this method, an antibody that completely inhibits the enzymatic activity of MM isozyme of creatine kinase and subunit M in MB isozyme without inhibiting the enzymatic activity of subunit B in creatine kinase MB isozyme prepared in advance is used. . That is, the test solution is first treated with the M-subunit inactivating antibody to limit the activity of creatine kinase to only that attributable to the creatine kinase MB isozyme. By measuring using reaction system 2, creatine kinase M
The B isozyme can be quantified.

[0011] In the previous clinical tests for creatine kinase and creatine kinase MB isozyme, a wet method in which a detection reaction is carried out in a reagent solution using the above-described method is generally used. Was. However, the quantitative analysis operation by the wet method has a disadvantage that it requires a considerably long time to obtain a result. That is, for example, for myocardial infarction, it is known that the shorter the time from the onset of infarction to the start of treatment, the higher the effect of the treatment is. high. Therefore, the long time from the start of the test to the acquisition of the test results
This may cause a delay in the start of treatment, which is undesirable.

As a means for obtaining a result of a clinical test in a short time, various dry analytical elements each having a structure in which a reagent layer containing a reagent composition involved in a detection reaction is laminated on a transparent support have been known. . A dry analytical element for quantifying creatine kinase and a dry analytical element for quantifying creatine kinase MB isozyme having a reagent layer containing a reagent composition involved in the above-mentioned creatine kinase test reaction or creatine kinase MB isozyme test reaction have already been developed. Has been used in practice. The configuration of such a dry analytical element for quantifying creatine kinase and the dry analytical element for quantifying creatine kinase MB isozyme are described in detail in the following patent publications. JP-A-61-254198, JP-A-61-2
No. 54199, Japanese Unexamined Patent Publication No. 61-260164,
JP-A-63-32499 mentioned above.

[0013] The above-mentioned dry analytical element for quantifying creatine kinase and the dry analytical element for quantifying creatine kinase MB isozyme are excellent ones which can be used for quantitative analysis of creatine kinase and creatine kinase MB isozyme in a short time, respectively. Although it is an analytical tool, there is a problem that its storage stability is poor from the production of an analytical element to its use in actual quantitative analysis. That is, it has been found that when each analytical element is stored under normal storage conditions, the sensitivity is reduced within a short period of time after production. Therefore, in order to maintain these dry analysis elements in a good storage state, it was necessary to freeze and store the analysis elements. For storage of such analytical elements,
The necessity of cryopreservation requires not only a facility for cryopreservation, but also a problem that a time delay is likely to occur when an actual clinical test is started.

[0014]

SUMMARY OF THE INVENTION An object of the present invention is to solve the low storage stability of a conventionally known dry analytical element for quantifying creatine kinase and a dry analytical element for quantifying creatine kinase MB isozyme. I do.
In particular, an object of the present invention is to provide a dry analytical element for quantifying creatine kinase and a dry analytical element for quantifying creatine kinase MB isozyme which can be stored at ordinary temperature and thus have a small decrease in sensitivity even when stored at ordinary temperature. I do.

[0015]

SUMMARY OF THE INVENTION The present invention provides a method for manufacturing a semiconductor device comprising the steps of:
Creatine phosphate and adenosine diphosphate to produce adenosine triphosphate in the presence of creatine kinase, and buffer in the pH 5.5-8.5 region to provide a pH environment for the production of adenosine triphosphate Glucose, hexokinase, nicotinamide adenine dinucleotide (phosphate) oxidized form, which contains a buffering compound exhibiting the ability to form a compound which can be detected by a spectrophotometric method by reacting with the produced adenosine triphosphate; and A dry analytical element for creatine kinase quantification obtained by laminating a creatine kinase detection reagent layer containing an indicator composition containing glucose-6-phosphate dehydrogenase, wherein creatine phosphate contains:
A dry analytical element for quantifying creatine kinase, which is arranged separately from adenosine diphosphate and glucose.

The present invention also provides creatine phosphate and adenosine diphosphate that form adenosine triphosphate in the presence of creatine kinase, and a pH environment for the production of the adenosine triphosphate. PH to provide
A glycerol, a glycerol kinase, which contains a buffering compound exhibiting a buffering capacity in the range of 5.5 to 8.5, and further produces a compound which can be detected by a spectrophotometric method by reacting with the produced adenosine triphosphate; L-α-
A dry analytical element for creatine kinase quantification obtained by laminating a creatine kinase detection reagent layer containing an indicator composition containing glycerophosphate oxidase and a leuco dye, wherein in the detection reagent layer, creatine phosphate is
There is also a dry analytical element for creatine kinase quantification characterized by being arranged separately from adenosine diphosphate and glycerol.

The present invention further provides an antibody, which specifically inhibits the creatine kinase M subunit activity, creatine kinase MB isozyme, on a support,
Creatine phosphate and adenosine diphosphate, which form adenosine triphosphate, and a buffering compound which exhibits a buffering capacity in a pH range of 5.5 to 8.5, which provides a pH environment for the production of adenosine triphosphate. Glucose, hexokinase, nicotinamide adenine dinucleotide (phosphate) oxidized form, and glucose-6-phosphate dehydrogenase which form a compound detectable by a spectrophotometric method by reacting with the generated adenosine triphosphate A creatine kinase MB isozyme quantification dry analytical element obtained by laminating a creatine kinase MB isozyme detection reagent layer containing an indicator composition containing, in the detection reagent layer, creatine phosphate is:
Creatine kinase M, which is arranged separately from adenosine diphosphate and glucose
There is also a dry analytical element for B isozyme quantification.

Further, the present invention provides a method for preparing a creatine kinase MB isozyme, which is an antibody that specifically inhibits the creatine kinase M subunit activity, on a support,
Creatine phosphate and adenosine diphosphate, which form adenosine triphosphate, and a buffering compound which exhibits a buffering capacity in a pH range of 5.5 to 8.5, which provides a pH environment for the production of adenosine triphosphate. Containing, further, glycerol which produces a compound detectable by a spectroscopic measurement method by reacting with the generated adenosine triphosphate,
A dry analytical element for quantifying creatine kinase MB isozyme comprising a creatine kinase MB isozyme detection reagent layer containing an indicator composition containing glycerol kinase, L-α-glycerophosphate oxidase and a leuco dye, wherein the detection reagent layer There is also a dry analytical element for quantifying creatine kinase MB isozyme, wherein creatine phosphate is disposed separately from adenosine diphosphate and glycerol.

In the dry analytical element for quantifying creatine kinase or the dry analytical element for quantifying creatine kinase MB of the present invention, when the detection reaction system 1 is used,
Creatine phosphate (CP) and adenosine diphosphate (ADP), which are reagent components involved in a reaction under the action of an enzyme, are separately arranged, and creatine phosphate and glucose (Glu) are separately arranged. It is characterized by: In the dry analytical element utilizing the detection reaction system 2, creatine phosphate (CP) and adenosine diphosphate (AD), which are also reagent components involved in the reaction under the action of an enzyme, are used.
P) and creatine phosphate and glycerol are separately disposed. In other words, in order to prevent or suppress the occurrence of side reactions during storage of the analytical element, two or more components involved in the enzymatic reaction in a series of detection reaction steps are separated and arranged, whereby the analytical element It is intended to improve the preservability of the product.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The detection reaction system used in the dry analytical element for quantifying creatine kinase or the dry analytical element for quantifying creatine kinase MB of the present invention is the above-mentioned detection reaction system 1 or detection reaction system 2. Therefore,
Any of the indicator compositions used in the above-described detection reaction system can be used as the indicator composition that produces a compound that can be detected by a spectroscopic measurement method by reacting with adenosine triphosphate. That is, examples of the indicator composition include the following indicator compositions.

1) An indicator composition containing glucose, hexokinase, nicotinamide adenine dinucleotide oxidized form, and glucose-6-phosphate dehydrogenase. 2) An indicator composition comprising glucose, hexokinase, nicotinamide adenine dinucleotide oxidized form, glucose-6-phosphate dehydrogenase and tetrazolium salt. 3) An indicator composition comprising glycerol, glycerol kinase, L-α-glycerophosphate oxidase and a leuco dye.

According to the study of the inventor of the present invention, the low storage stability of the conventional dry analytical element for quantifying creatine kinase and the conventional dry analytical element for quantifying creatine kinase MB isozyme is due to the fact that the reagent composition has a low It was found that the reagent in the reaction was caused by a gradual reaction in the absence of creatine kinase (CK) or creatine kinase MB isozyme (CKMB). That is, in the detection reaction system 1 described above, CK or CKM
Even if B does not exist, CP and ADP partially react to form ATP, and CP and Glu react to form G6.
The phenomenon that P was generated was confirmed. On the other hand, in the detection reaction system 2, in addition to the production of ATP due to the partial reaction between CP and ADP, the production of L-α-glycerophosphate due to the reaction between CP and glycerol was also confirmed. Was done. Therefore, a reaction that occurs in the absence of these creatine kinases or creatine kinase MB isozymes results in a significant decrease in analytical accuracy.

For the above reasons, the present inventor has studied with the aim of suppressing the undesirable side reactions that occur during storage of the dry analytical element at room temperature as described above. As a result, as described above, it is possible to prevent or suppress the generation of the side reaction by separately arranging the components involved in the enzymatic reaction in the series of detection reactions in the analytical element. And arrived at the present invention.

The constitution of the analytical element of the present invention, that is, the dry analytical element for quantifying creatine kinase or the dry analytical element for quantifying creatine kinase MB isozyme, and various reagents constituting them are already known. In the dry analytical element described above, the same various reagents having the same configuration are used except that the above-mentioned specific buffer compound is used. The configuration of such a dry analytical element and examples of reagents are described in detail in the above-mentioned patent publications and publications, and will not be described again here.

The reaction between phosphoric acid (CP) and adenosine diphosphate (ADP) for producing adenosine triphosphate (ATP) by the enzymatic action of creatine kinase or creatine kinase MB isozyme requires a specific pH.
It has been known for some time that an environment is required, i.e. a pH environment of pH 5.5 to 8.5, and for this reason Good Buffer, abbreviated as Bis-Tris in its early days, The composition was used, but then imidazole is commonly used as a buffering compound according to the JSCC recommendations for quantification of creatine kinase.

However, according to the research of the present inventors, the buffering agent used for the above purpose is selected from buffering compounds having a sulfonic acid group, particularly buffering compounds generally called Good's buffering agents. It has been found that a buffering compound having a sulfonic acid group is preferred. That is, Good's buffers include those having a sulfonic acid group and those without a sulfonic acid group,
The buffering compound used in the present invention is a buffering compound having a sulfonic acid group among them. The Good's buffer (also called Good's buffer) is described in, for example, “Chemical Dictionary” (Morikita Shuppan Co., Ltd., 198).
Published one year), "Chemical Encyclopedia" (Tokyo Chemical Doujin Inc.,
1989) and further detailed descriptions in many books on clinical test reagents.

The buffering compounds belonging to Good's buffer and suitable for use in the dry analytical element of the present invention include:
TES, TAPSO, MOPSO, MES, DIPS
Compounds named O, HEPES, HEPSO can be mentioned. Among these compounds, TE
Compounds named S, TAPSO, MOPSO, and MES are preferred. Particularly preferred is TES
And TAPSO.

In the present invention, the separation of the chemical components used in the detection reaction systems 1 and 2 can be performed by detecting the detection reagent layer of the analytical element (as long as the detection reagent is contained, a developing layer, an adhesive layer, And all the layers having the function of (i.e., a layer having the function of) are formed, and two or more layers (reagent layers) are formed, and the chemical components involved in the enzyme reaction system for detection are separated into the reagent layers according to the present invention. And place them. Alternatively, a method of avoiding physical contact between chemical components during storage of the analysis element using a microcapsule or the like that can penetrate moisture can be used.

Creatine kinase (CK) or creatine kinase MB isozyme (CKM) using a dry analytical element for quantifying creatine kinase or a dry analytical element for quantifying creatine kinase MB isozyme
The operation of the quantitative analysis of B) is already known as described in the above-mentioned patent publications and patent publications,
CK or CKMB using the dry analytical element of the present invention
Can be performed according to the same operation as the above.

Next, examples of the present invention and comparative examples will be described. In the following Examples and Comparative Examples, only a dry analytical element for quantifying creatine kinase MB isozyme using the detection reaction systems 1 and 2 is shown, but the function of the buffering compound used in the present invention is as follows. , Creatine kinase MB
There is no substantial difference between the case for isozyme quantification and the case for creatine kinase (total creatine kinase) quantification. Therefore, according to the following example,
It should be understood that the effect of separating the chemical components involved in the enzymatic reaction used in each detection reaction system of the present invention has been clarified in any of the dry analysis elements of the above embodiments.

[0031]

【Example】

[Example 1] CK according to the present invention using detection reaction system 1
Production of analytical element for MB quantification Transparent polyethylene terephthalate film (thickness 180
μm) was subjected to a hydrophilic treatment, and a coating solution having the following composition was applied on the treated surface and dried to form a reagent layer (a layer containing an indicator and CK) having a dry layer thickness of 12 μm.

Deionized gelatin 200 g Water 1040 g 5% nonionic surfactant aqueous solution 80 g Nitrotetrazolium blue 10 g Diaphorase 69230U Creatine phosphate (CK) 60 g

A tricot knit made of spun polyethylene tetraphthalate yarn prepared separately was impregnated with a coating solution having the following composition at a coating amount of 100 g / m ² and dried. Then, an adhesive was applied to the surface of the reagent layer in the form of a halftone dot, and the coated tricot knit was pressure-bonded onto the adhesive to integrate the adhesive.

36.6 g of water 20 g of anti-human CK-M goat antibody solution (solution having an ability to inhibit 50% or more of 2,000 U / L CKMM at 1500 times final dilution) 10% nonionic surfactant aqueous solution 9 g 15% aqueous polyacrylamide solution 13.8 g (average molecular weight 37,000) 20% magnesium chloride aqueous solution 8.7 g ethylenediaminetetraacetic acid / 2Na salt 0.5 g glucose 0.62 g adenosine-5'-diphosphate (ADP) 3g P ^1, P ⁵ - di (adenosine-5') penta phosphate (AP5A) 0.25 g adenosine 5'-monophosphate (AMP) 1.2 g N-acetyl -L- cysteine 0.1g nicotinamide adenine nucleotides Oxidized form (NAD) 0.6 g Imidazole 1.5 g 1N HCl 1 0.0 g glucose-6-phosphate dehydrogenase 14000 U hexokinase (HK) 19400 U ascorbate oxidase 5000 U

Lastly, the above-formed laminate in which two reagent layers (a layer containing an indicator and CK and a developing layer containing another reaction reagent) are laminated on a support is 12 m in length.
By cutting into dimensions of mx 12 mm, an analytical element for quantifying CKMB having a plurality of reagent layers according to the present invention was obtained.

[Comparative Example 1] Production of an analytical element for CKMB quantification in which a detection reaction component was arranged in one layer A transparent polyethylene terephthalate film (thickness: 180)
μm) was subjected to a hydrophilic treatment, and a coating solution having the following composition was applied on the treated surface and dried to form an indicator layer having a dry layer thickness of 12 μm.

Deionized gelatin 200 g Water 1100 g 5% nonionic surfactant aqueous solution 80 g Nitrotetrazolium blue 10 g Diaphorase 69230U

After the indicator layer is moistened with a solution containing a gelatin crosslinking agent (about 30 g / m ² ), a tricot knit made of a spun yarn of polyethylene tetraphthalate is pressed on the indicator layer, dried and developed. Layers. Next, a coating solution having the following composition was applied on the surface of the spread layer at a coating amount of 130 g / m ² and dried.

46.6 g of water 20 g of anti-human CK-M goat antibody solution (solution capable of inhibiting 2000 U / L of CKMM by 50% or more at a final dilution of 1500 times) 10% nonionic surfactant aqueous solution 9 g 15% aqueous solution of polyacrylamide 33.8 g (average molecular weight 37,000) 20% aqueous magnesium chloride solution 8.7 g ethylenediaminetetraacetic acid / 2Na salt 0.5 g creatine phosphate (CK) 1.3 g adenosine-5'-diphosphate ^{(ADP) 0.3g P 1, P} 5 - di (adenosine-5') penta phosphate (AP5A) 0.25 g adenosine 5'-monophosphate (AMP) 1.2 g N-acetyl -L- cysteine 0. 1 g nicotinamide adenine nucleotide oxidized form (NAD) 0.6 g imidazole 1.5 g N HCl 10.0 g glucose 0.62g glucose-6-phosphate dehydrogenase 14000U hexokinase (HK) 19400U ascorbate oxidase 5000U

Lastly, the laminate formed above, in which the indicator layer and the reaction reagent-containing developing layer were laminated on the support, was
This was cut into a size of 2 mm × 12 mm to obtain a comparative CKMB quantitative analysis element in which the detection reaction reagent was contained in the same reagent layer.

[Evaluation of analytical element for quantification of CKMB: generation of ATP during storage] The analytical elements obtained in Example 1 and Comparative Example 1 were subjected to storage under conditions of 35 ° C. and 11% RH, respectively.
After storing for 0 day, a trace amount of water was spotted on the surface of the developing layer of each analytical element and incubated at 37 ° C. for 6 minutes. Next, at a wavelength of 540 nm, the coloration generated in each analytical element was measured. In parallel, Example 1 immediately after manufacturing
The same color test was performed for the analysis elements of Comparative Example 1 and Comparative Example 1. The coloration of each analytical element found by this measurement is shown in Table 1 below.

[0042]

[Table 1] Table 1 分析 Analytical element Detection reagent color ( Arrangement of optical density: OD) Analytical element immediately after production Analytical element stored for 1 day ───────────────────────────────── Example 1 Separation arrangement 0.35 0.36 Comparative example 1 Arrangement in the same layer 0.36 0.48 ─────────────

From the results shown in Table 1 above, in the analytical element in which the detection reagents involved in the enzyme reaction used in the present invention were separated and arranged, the ATP in the absence of creatine kinase MB isozyme was It can be seen that by-products are effectively suppressed.

[Evaluation of CKMB Quantitative Analytical Element: Sensitivity Fluctuation During Storage] Analytical element obtained in Example and Comparative Example 1
For each of the obtained analytical elements, one immediately after production and one after storage for 10 days under the storage conditions of 35 ° C. and 11 RH% were prepared, and the CKMB content of each analytical element was about 5 U / L. Sample, CKMB content about 15U / L, and CKMB content about 280U
/ L samples were separately spotted at three levels. The analytical element on which the sample was spotted was incubated at 37 ° C., and then subjected to spectrometry at a wavelength of 540 nm at 2 minutes and 5 minutes after the start of the incubation, and referred to a calibration curve prepared in advance. Then, the amount of CKMB was determined by the reaction rate method. The results are shown in Table 2 below.

[0045]

[Table 2] Table 2 分析 Analysis element Sample CKMB measurement value ( (CKMB amount) Analytical element immediately after production Analytical element after storage for 10 days 日Example 1 About 5U / L 5U / L 5U / L About 15U / L 15U / L 12U / L About 280U / L 280U / L 275U / L ──────────────────── Comparative Example 1 About 5U / L 5U / L 25U / L About 15U / L 15U / L 40U / L About 280U / L 280U / L 295U / L ────────────────────────────────────

From the results in Table 2 above, it can be seen that the CKM of the present invention
When the analytical element for B quantification (Example 1) is used, it can be seen that the variation in sensitivity regarding the CKMB measurement is slight even under more severe storage conditions than normal storage conditions. on the other hand,
The conventional analytical element for CKMB quantification (Comparative Example 1) shows satisfactory sensitivity immediately after its production, but the sensitivity is clearly lowered by storage (generation of background color by ATP by-product). Appears.

[Example 2] Production of analytical element for quantitative determination of CKMB of the present invention using detection reaction system 2 A transparent polyethylene terephthalate film (thickness: 180)
μm) was subjected to a hydrophilic treatment, and a coating solution having the following composition was applied on the treated surface and dried to form a reagent layer (a layer containing an indicator and CK) having a dry layer thickness of 12 μm.

Deionized gelatin 200 g Water 930 g Nonionic surfactant 6.5 g Methylene blue leuco 3.5 g Methanol 190 g Creatine phosphate (CK) 60 g Peroxidase 175000 U

A separately prepared tricot knitted yarn made of polyethylene tetraphthalate spun yarn was impregnated with a coating solution having the following composition at a coating amount of 100 g / m ² and dried. Then, an adhesive was applied to the surface of the reagent layer in the form of a halftone dot, and the coated tricot knit was pressure-bonded onto the adhesive to integrate the adhesive.

36.6 g of water 20 g of an anti-human CK-M goat antibody solution (a solution capable of inhibiting 50% or more of 2,000 U / L CKMM at a final dilution of 1500 times) 10% aqueous solution of a nonionic surfactant 9 g 15% polyacrylamide aqueous solution 13.8 g (average molecular weight 37,000) 20% magnesium chloride aqueous solution 8.7 g ethylenediaminetetraacetic acid / 2Na salt 0.5 g glycerol 0.31 g adenosine-5'-diphosphate (ADP) 3g P ^1, P ⁵ - di (adenosine-5') penta phosphate (AP5A) 0.25 g adenosine 5'-monophosphate (AMP) 1.2 g N-acetyl -L- cysteine 0.1g imidazole 1.5g 1N HCl 10.0 g glycerol kinase 12000 U L-α-glycero Phosphate oxidase 6500U ascorbic acid oxidase 5000U

Lastly, the laminated body formed as described above, in which two reagent layers (a layer containing an indicator and CK and a developing layer containing another reaction reagent) are laminated on a support, is 12 m in length.
By cutting into dimensions of mx 12 mm, an analytical element for quantifying CKMB having a plurality of reagent layers according to the present invention was obtained.

[Comparative Example 2] Production of analytical element for CKMB quantification in which detection reaction components were arranged in one layer A transparent polyethylene terephthalate film (thickness: 180)
μm) was subjected to a hydrophilic treatment, and a coating solution having the following composition was applied on the treated surface and dried to form an indicator layer having a dry layer thickness of 12 μm.

Deionized gelatin 200 g Water 930 g Nonionic surfactant 6.5 g Methylene blue leuco 3.5 g Methanol 190 g Peroxidase 175000 U

After the indicator layer was moistened with a solution containing a gelatin cross-linking agent (about 30 g / m ² ), a tricot knit made of a spun yarn of polyethylene tetraphthalate was pressed on the indicator layer, dried and developed. Layers. Next, a coating solution having the following composition was applied on the surface of the spread layer at a coating amount of 130 g / m ² and dried.

46.6 g of water 20 g of anti-human CK-M goat antibody solution (solution having an ability to inhibit 50% or more of 2,000 U / L CKMM at a final dilution of 1500 times) 10% nonionic surfactant aqueous solution 9 g 15% aqueous solution of polyacrylamide 33.8 g (average molecular weight 37,000) 20% aqueous solution of magnesium chloride 8.7 g ethylenediaminetetraacetic acid / 2Na salt 0.5 g creatine phosphate (CK) 1.3 g glycerol 0.31 g adenosine-5 '- diphosphate ^{(ADP) 0.3g P 1, P} 5 - di (adenosine-5') penta phosphate (AP5A) 0.25 g adenosine 5'-monophosphate (AMP) 1.2 g N-acetyl -L -Cysteine 0.1 g imidazole 1.5 g 1N HCl 10.0 g glycerol quina 12,000 U L-α-glycerophosphate oxidase 6500 U Ascorbate oxidase 5000 U

Finally, the laminated body formed above was
The sample was cut into a size of mm × 12 mm to obtain a comparative CKMB quantitative analysis element. [Evaluation of analytical element for CKMB quantification: generation of ATP during storage] As a result of evaluating the analytical elements obtained in Example 2 and Comparative Example 2 by the above-mentioned method, the same tendency as the above-mentioned results appears. It could be confirmed.

[Evaluation of analytical element for CKMB quantification: sensitivity fluctuation during storage] As a result of evaluating the analytical elements obtained in Example 2 and Comparative Example 2 by the above-mentioned method, the same tendency as the above-mentioned results appears. That was confirmed.

[0058]

Industrial Applicability According to the analytical element of the present invention in which reagent components involved in an enzymatic reaction used for quantitative analysis of creatine kinase (CK) or creatine kinase MB isozyme (CKMB) are arranged separately from each other, storage stability is remarkable. Therefore, it is not necessary to preserve the analytical element in a frozen state, which has been indispensable at the time of storage before use of the conventional dry analytical element for quantitative analysis of CK or CKMB, which is very advantageous in practical use.

Claims (10)

[Claims] 1. A creatine phosphate and adenosine diphosphate that produce adenosine triphosphate in the presence of creatine kinase on a support, and a pH5 that provides a pH environment for the production of adenosine triphosphate. 0.5-8.5
Glucose, hexokinase, nicotinamide adenine dinucleotide (phosphate) which contains a buffering compound exhibiting a buffering capacity in the region of A) a dry analytical element for creatine kinase quantification comprising a creatine kinase detection reagent layer containing an indicator composition containing an oxidized form and glucose-6-phosphate dehydrogenase, wherein creatine phosphate is contained in the detection reagent layer. Is disposed separately from adenosine diphosphate and glucose, and a dry analytical element for creatine kinase quantification. 2. The dry analytical element for creatine kinase determination according to claim 1, wherein the indicator composition further comprises a tetrazolium salt. 3. The creatine kinase detection reagent layer comprises at least two reagent layers, one of which contains creatine phosphate and the other contains adenosine diphosphate and glucose. The dry analytical element for creatine kinase quantification according to claim 1 or 2. 4. On a support, creatine phosphate and adenosine diphosphate, which produce adenosine triphosphate in the presence of creatine kinase, and pH 5, which provides a pH environment for the production of adenosine triphosphate. 0.5-8.5
Glycerol, glycerol kinase, L-α-glycerophosphate oxidase, which contains a buffering compound exhibiting a buffering capacity in the region, and further forms a compound detectable by a spectroscopic measurement method by reacting with the generated adenosine triphosphate. And a creatine kinase detection reagent layer comprising a creatine kinase detection reagent layer containing an indicator composition containing a leuco dye and a leuco dye, wherein creatine phosphate contains adenosine diphosphate and glycerol in the detection reagent layer. 1. A dry analytical element for quantifying creatine kinase, wherein the analytical element is separated from the element. 5. The creatine kinase detection reagent layer comprises at least two reagent layers, one of which contains creatine phosphate and the other reagent layer contains adenosine diphosphate and glycerol. The dry analytical element for creatine kinase quantification according to claim 4. 6. An antibody that specifically inhibits the M subunit activity of creatine kinase, creatine phosphate and adenosine diphosphate that generate adenosine triphosphate in the presence of a creatine kinase MB isozyme,
It contains a buffering compound exhibiting a buffering capacity in a pH range of 5.5 to 8.5, which provides a pH environment for the production of adenosine triphosphate, and is further subjected to spectroscopy by reaction with the generated adenosine triphosphate. Kinase MB isozyme detection reagent layer containing an indicator composition containing glucose, hexokinase, nicotinamide adenine dinucleotide (phosphate) oxidized form, and glucose-6-phosphate dehydrogenase, which produces a compound detectable by a quantitative measurement method. Creatine kinase MB composed of layers
A dry analytical element for isozyme quantification, characterized in that creatine phosphate is arranged separately from adenosine diphosphate and glucose in the detection reagent layer. element. 7. The dry analytical element for quantifying creatine kinase MB isozyme according to claim 6, wherein the indicator composition further comprises a tetrazolium salt. 8. The creatine kinase MB isozyme detection reagent layer comprises at least two reagent layers, one of which contains creatine phosphate, and the other contains adenosine diphosphate and glucose. The dry analytical element for creatine kinase MB isozyme quantification according to claim 6 or 7, wherein 9. An antibody that specifically inhibits the creatine kinase M subunit activity, creatine phosphate and adenosine diphosphate, which produce adenosine triphosphate in the presence of a creatine kinase MB isozyme,
It contains a buffering compound exhibiting a buffering capacity in a pH range of 5.5 to 8.5, which provides a pH environment for the production of adenosine triphosphate, and is further subjected to spectroscopy by reaction with the generated adenosine triphosphate. Kinase MB isozyme comprising a creatine kinase MB isozyme detection reagent layer containing an indicator composition containing glycerol, glycerol kinase, L-α-glycerophosphate oxidase and a leuco dye, which produces a compound detectable by a quantitative measurement method A dry analytical element for quantification, wherein creatine phosphate is disposed separately from adenosine diphosphate and glycerol in the detection reagent layer.
Dry analytical element for quantification of B isozyme. 10. The creatine kinase MB isozyme detection reagent layer comprises at least two reagent layers, one of which contains creatine phosphate, and the other contains adenosine diphosphate and glycerol. The dry analytical element for quantifying creatine kinase MB isozyme according to claim 9.