JP2005114368A - Enzyme stabilizing method and specimen analyzing tool used therein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a specimen analyzing tool capable of preventing the deactivation of enzyme in a dry state. <P>SOLUTION: The specimen analyzing is manufactured by impregnating a porous material with a solution containing enzyme taking a component to be analyzed as a substrate and a protein hydrolysate and drying the impregnated porous material. As the protein hydrolysate, a gelatin hydrolysate can be used and, as the porous material, filter paper made of cellulose can be used. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an enzyme stabilization method for preventing enzyme deactivation in a dry state and a sample analysis tool used therefor.

  Conventionally, when a biological sample such as blood or urine is examined, a specimen analysis tool is widely used which performs analysis by impregnating the sample with a porous material. In the sample analysis tool, usually, various reagents such as enzymes using the analysis target component in the sample as a substrate are held in advance in the porous material.For example, by impregnating the porous material with the sample, The reagent in the porous material can be reacted with the component to be analyzed in the sample, and the reaction result can be directly measured by an optical technique or an electrochemical technique.

  Such a sample analysis tool is used in general clinical tests and the like, but in recent years, it has been further studied and actually used for a remote clinical test system. The remote clinical test system is a system in which a patient or the like collects blood at home, impregnates and dries the blood test tool, and mails it to a test organization such as a hospital for testing. According to this system, patients can know the test results by mail without going to the hospital, and can go to the hospital to receive treatment when the test results come out. Blood collection is also unnecessary, and labor on both the hospital side and the patient side is reduced.

In general, the sample analysis tool can be manufactured by impregnating the porous material in a reagent solution and drying it. However, there is a problem that the enzyme retained by drying the porous material is deactivated, and the enzyme activity further decreases during storage of the sample analysis tool. For this reason, it is necessary to make the amount of enzyme retained in the porous material excessive, and there is a problem in terms of cost. In order to solve such problems, there is a method in which proteins such as bovine serum albumin, polysaccharides such as sodium alginate, trehalose, and the like are allowed to coexist as stabilizers (for example, Patent Document 1).
JP-T 63-500562

  However, such stabilizers have the following problems. That is, since many of the stabilizers generally have a polymer structure, it is difficult to dissolve in an aqueous solvent and the viscosity of the solution itself is high, so that it is difficult to impregnate the porous material. Even when a sample is actually added, the reaction rate is slow because it takes time to dissolve the sample. Furthermore, as the effect, a remarkable effect cannot be expected for the polysaccharide such as sodium alginate. On the other hand, bovine serum albumin is expensive, and cheap egg albumin is difficult to use because it contains insoluble components. In addition, since urine test paper uses protein as an analysis item, it is difficult to use such a protein as a stabilizer.

  Accordingly, an object of the present invention is to provide a method for stabilizing an enzyme and a sample analysis tool used therefor, which are inexpensive and effectively prevent the enzyme from being deactivated in a dry state.

  In order to achieve the above object, the stabilization method of the present invention is a method of stabilizing an enzyme in a dry state, wherein the enzyme is allowed to coexist with a proteolysate.

  The sample analysis tool of the present invention is a sample analysis tool in which an enzyme is held in a porous material, wherein the porous material further holds a protein degradation product, Can be used for stabilization method.

  According to such a stabilization method and sample analysis tool of the present invention, inactivation of the enzyme can be suppressed by allowing the enzyme to coexist with a proteolysate in a dry state. For this reason, the usage-amount of the said enzyme can be reduced, for example, the expiration date of a sample analysis tool can fully be extended, and cost reduction can be achieved. In addition, even if the sample analysis tool is used for urine testing, it uses a protein degradation product as a stabilizer, so even if the analysis item is protein, it may affect the analysis accuracy. Absent. Furthermore, when preparing a sample analysis tool, there are no problems such as viscosity, solubility, and transparency as in the case of conventional stabilizers, so it is very easy to handle, the manufacturing process is simplified, and the enzyme by drying treatment is simplified. Deactivation can also be prevented.

  In the stabilization method and the sample analysis tool of the present invention, the protein degradation product preferably contains a polypeptide having a molecular weight of 500 to 10,000, more preferably a polypeptide having a molecular weight of 1000 to 5000. The protein degradation product includes a denatured protein degradation product. As the protein degradation product, specifically, gelatin hydrolyzate, casein hydrolyzate, soybean hydrolyzate and the like can be used, and among them, gelatin degradation product is preferable. Such a protein degradation product may be prepared by, for example, degrading a protein with various proteases, or a commercially available product may be used. As the gelatin degradation product, for example, trade names NIPPEPEPTIDE PA-10, PE-20, PA-100 and AFC (manufactured by Nippi) can be used.

  In the present invention, the enzyme is appropriately selected according to the component to be analyzed. However, since the present invention is useful for an enzyme in which inactivation in a dry state is a problem, for example, glucose oxidase (GOD), peroxidase (POD), ascorbate oxidase (AsOD), hexokinase, diaphorase, pyruvate oxidase, cholesterol oxidase (COD) and the like are preferable. Specifically, when the analysis target component is glucose, for example, a combination of GOD and POD is preferable, and when the analysis target component is cholesterol, for example, a combination of COD and POD is preferable.

  The ratio of the protein degradation product added to the enzyme can be determined as appropriate according to, for example, the type of enzyme, and is, for example, in the range of 0.8 to 20,000 mg of the protein degradation product per 1 KU of enzyme. Is preferable, more preferably in the range of 50 to 2000 mg, and particularly preferably in the range of 200 to 1000 mg.

  Next, the stabilization method of the present invention will be described. The stabilization method of the present invention can be carried out, for example, by preparing a mixed solution in which the enzyme and the protein degradation product are dissolved in an aqueous solvent and drying the mixed solution. Specifically, it is preferable that the enzyme and the protein degradation product are allowed to coexist in a dry state by impregnating a porous material with a solution containing the enzyme and the protein degradation product and drying the porous material. The porous material thus prepared is the sample analysis tool of the present invention holding the enzyme and the protein degradation product. As described above, inactivation of the enzyme due to the drying treatment can be prevented by subjecting the porous material to a drying treatment in the presence of the enzyme and the protein degradation product.

  The enzyme concentration in the mixed solution is, for example, in the range of 5 to 2000 KU / L, preferably in the range of 50 to 1000 KU / L, and more preferably in the range of 100 to 500 KU / L. Moreover, the proteolysate concentration in the mixed solution is, for example, in the range of 5 to 200 g / L, preferably in the range of 10 to 100 g / L, and more preferably in the range of 20 to 50 g / L. In the present invention, since the protein degradation product is used, the mixed solution to be prepared is not in a suspended state but has excellent transparency in which the protein degradation product has been sufficiently dissolved. It is excellent in handling for the production of analytical tools.

  The pH of the mixed solution is, for example, in the range of 2 to 10, and preferably 4 to 9. Moreover, water, various buffer solutions, etc. can be used as said aqueous solvent.

The amount of the mixed solution impregnated into the porous material is not particularly limited, and can be appropriately determined according to the amount of the enzyme to be retained in the porous material as described later. For example, per 1 cm ^{3 of the} porous material, for example, 0.1 to 1.5 mL, preferably 0.3 to 1.2 mL, and more preferably 0.5 to 1.0 mL. In addition, in this invention, the volume (cm < ³ >) of a porous material is a volume containing a void | hole part.

  The porous material is not particularly limited, but filter paper is preferable, and cellulose is preferable as the material. In particular, when GOD is impregnated into cellulose filter paper, there is a problem that GOD deteriorates due to the reaction of GOD with glucose at the end of cellulose. Although it is unknown, there is an effect that the reaction between the two can be suppressed and the deactivation of GOD can be prevented. As such an effect, a gelatin hydrolyzate as described above is particularly preferable.

  In the sample analysis tool of the present invention, the amount of the enzyme to be held in the porous material can be appropriately determined according to, for example, the type of enzyme. According to the present invention, since the amount of enzyme to be used can be reduced as described above, it is possible to reduce the amount to 1/2 to 1/5 of the conventional sample analysis tool. In particular, when glucose is a component to be analyzed, the amount of GOD and POD can be reduced to 1/2 to 1/3 of the conventional amount.

On the other hand, the amount of the protein degradation product retained in the porous material is, for example, 5 to 200 mg, preferably 10 to 100 mg, more preferably 20 to 50 mg per 1 cm ^{3 of the} porous material.

  Further, in the sample analysis tool of the present invention, a chromogenic substrate that develops color by oxidation may coexist in a dry state. If the chromogenic substrate is colored with the reaction between the various enzymes and the analyte as described above, the presence or concentration of the analyte is analyzed by confirming the degree of color development by visual observation or absorbance measurement. Because it can. The chromogenic substrate can be appropriately determined according to, for example, the type of component to be analyzed and the type of enzyme used, and conventionally known ones can be used. Specifically, when the analysis target component is glucose and GOD and POD are used as enzymes, hydrogen peroxide is generated by the reaction of glucose and GOD, and this hydrogen peroxide is reacted with POD. Accordingly, a substrate that develops color by oxidation is preferable, and for example, Trinder reagent, rotolycin, TMBZ, and the like can be used.

  Example 1 is an example in which a sample analysis tool in which a proteolytic product is added to various enzymes is prepared, and suppression of inactivation of the enzyme is confirmed.

  GOD solution, POD solution and AsOD solution are prepared to have the following composition, and gelatin hydrolyzate (trade name Nippi Peptide PA-10; manufactured by Nippi Co., Ltd., the same applies below) as a protein degradation product in 10 mL of these enzyme solutions Were dissolved to prepare mixed solutions, respectively. In this mixed solution, 8 mm × 27 mm filter paper (trade name: 3MMCHR; manufactured by Whatman Co., Ltd., hereinafter the same) was immersed, and the mixed solution was sufficiently infiltrated into the filter paper. And after removing the excess mixed solution, the drying process was performed for 30 minutes using the ventilation drying machine (40 degreeC), and the porous material which hold | maintained the enzyme was prepared. As a stabilizer, instead of the gelatin hydrolyzate, soybean peptone (manufactured by Nacalai Tesque), casein peptone (manufactured by Nacalai Tesque), bovine serum albumin (manufactured by Wako Pure Chemical Industries), sodium glutamate (Nacalai Tesque) ), Trehalose (manufactured by Wako Pure Chemical Industries, Ltd.), α-cyclodextrin (manufactured by Nacalai Tesque), and reduced glutathione (manufactured by Nacalai Tesque) were used in the same manner except that 500 mg each was used as Reference Example 1. . Further, Comparative Example 1 was prepared in the same manner except that no stabilizer was added.

(GOD solution)
0.1M MES buffer (pH 5.7) 100mL
GOD (Toyobo Co., Ltd.) 10KU
(POD solution)
0.1M MES buffer (pH 5.7) 100mL
POD (Toyobo) 10KU
(AsOD solution)
0.1M MES buffer (pH 5.7) 100mL
AsOD (Toyobo) 7KU
Residual activity of the enzyme was confirmed using the porous material holding the enzyme prepared as described above as a sample. First, five sample pieces each having a diameter of 6 mm were cut out from each sample using a punch. These sample pieces were immersed in 10 mL of 0.1 M MES buffer (pH 5.7) and allowed to stand at room temperature for 60 minutes to extract the enzyme. In addition, it has been confirmed separately that no enzyme remains in the filter paper in the case of extraction for 60 minutes. Then, the enzyme activity is measured for each eluate obtained by the extraction treatment, the total enzyme activity amount (U) of the eluted enzyme is obtained, and the activity (U / L) is also obtained for the prepared mixed solution. It was. The activities of GOD, POD and AsOD were performed according to the activity measurement method of the manufacturer of the enzyme used.

  On the other hand, in order to determine the residual activity, a dye (trade name Blue No. 1; manufactured by Tokyo Chemical Industry Co., Ltd.) was further dissolved in the mixed solution prepared in the same manner as described above so as to be 1 mM, and 8 mm × 27 mm was dissolved in this solution. The filter paper was dipped, dried, cut out and extracted in the same manner, and the amount of impregnation of the mixed solution per volume of the porous material was determined.

Then, from the impregnation amount and the activity (U / L) of the mixed solution, the amount of enzyme in the porous material before the drying treatment is obtained, and when this value is taken as 100%, the remaining activity after the drying treatment (% ) The results are shown in Tables 1 to 3 below. Table 1 below shows the results of GOD, Table 2 below shows the results of POD, and Table 3 below shows the results of AsOD.
(Table 1)
GOD stabilizer Residual activity (%)
Comparative Example 1 No addition 16.9
Example 1 Gelatin hydrolyzate 50.4
Reference Example 1 Soybean peptone 43.4
Casein peptone 51.6
Bovine serum albumin 73.5
Sodium glutamate 40.8
Trehalose 42.8
α-Cyclodextrin 33.1
Reduced glutathione 33.9

(Table 2)
POD stabilizer Residual activity (%)
Comparative Example 1 No additive 46.6
Example 1 Gelatin hydrolyzate 78.1
Reference Example 1 Soybean peptone 58.4
Casein Peptone 75.0
Bovine serum albumin 76.7
Sodium glutamate 69.3
Trehalose 69.0
α-Cyclodextrin 69.2
Reduced glutathione 46.6

(Table 3)
AsOD Stabilizer Residual activity (%)
Comparative Example 1 No addition 27.7
Example 1 Gelatin hydrolyzate 46.0
Reference Example 1 Soybean peptone 35.2
Casein peptone 39.7
Bovine serum albumin 39.4
Sodium glutamate 31.7
Trehalose 34.9
α-Cyclodextrin 41.3
Reduced glutathione 31.9

As shown in Tables 1 to 3, it can be seen that by allowing the gelatin hydrolyzate to coexist in a dry state, inactivation of the enzyme could be remarkably suppressed as compared with the comparative example in which no stabilizer was added. Moreover, the deactivation suppression ability with respect to POD and AsOD showed the result superior to other stabilizers.

(Reference Example 2)
50 μL of a mixed solution containing no gelatin hydrolyzate prepared in the same manner as in Comparative Example 1 was dropped onto a glass watch glass, dried at 40 ° C. for 120 minutes, and further added to a 0.1 M MES buffer ( pH 5.7) 1000 μL was added dropwise to dissolve the enzyme, and the activity of this solution was measured. And the residual activity (%) when activity in the prepared mixed solution was made into 100% was calculated | required. The results are shown in Table 4 below together with the results in Comparative Example 1. From the results shown in Table 4 below, it can be seen that the enzyme activity is extremely lowered by impregnating a porous material with these enzymes and drying.
(Table 4)
Enzyme Residual activity (%)
Reference Example 2 GOD 95.8
Comparative Example 1 GOD 16.9
Reference Example 2 POD 95.6
Comparative Example 1 POD 46.6
Reference Example 2 AsOD 73.5
Comparative Example 1 AsOD 27.7

A GOD solution, a POD solution, and an AsOD solution are prepared so as to have the composition shown in Example 1, and a predetermined amount (100, 300, 500, 1000 mg) of gelatin hydrolyzate as a protein degradation product is further added to 10 mL of these enzyme solutions. ) Dissolved to prepare mixed solutions respectively (stabilizer concentrations 1, 3, 5, 10%). Using these mixed solutions, a porous material sample holding an enzyme was prepared in the same manner as in Example 1, and the residual activity (%) was determined in the same manner as in Example 1. Moreover, except not adding a stabilizer, what was prepared similarly was made into the comparative example 2, and residual activity (%) was calculated | required similarly. The results are shown in Table 5 below.
(Table 5)
Stabilizer GOD residual activity POD residual activity AsOD residual activity
(%) (%) (%)
Comparative Example 2 0% 16.9 46.6 27.7
Example 2 1% 34.1 60.2 35.6
3% 48.6 75.9 42.2
5% 50.4 78.1 46.0
10% 52.0 83.3 49.3

As shown in Table 5, the inactivation of the enzyme could be further suppressed by increasing the amount of gelatin hydrolyzate.

  In this example, suppression of deactivation of GOD and POD was confirmed for a glucose test piece obtained by impregnating and drying a porous material with GOD, POD and color former in the presence of a protein degradation product.

  A mixed solution was prepared so as to have the following composition. In addition, the addition amount of GOD and POD was changed in the following mixed solution. That is. The amount of POD added when the GOD is set to 20 KU is “10, 8, 6, 4, 2, 1 KU / 100 mL”, and the GOD when the POD is set to 10 KU is “20, 16, 12, 8, 4”. 2KU / 100mL ". In these mixed solutions, 30 cm × 20 cm filter paper (trade name: 3MMCHR; manufactured by Whatman Co., Ltd., hereinafter the same) was immersed, and the mixed solution was sufficiently infiltrated into the filter paper. And after removing the excess mixed solution, the drying process was performed for 30 minutes using the ventilation drying machine (40 degreeC), and the porous material which hold | maintained the enzyme was prepared. A double-sided tape was affixed to one surface of the porous material and cut into a size of 5 mm × 5 mm. This slice was attached to the tip of a white polyethylene terephthalate (PET) film (thickness 250 μm, 5 mm × 70 mm) via the double-sided tape to obtain a glucose test piece.

  Then, the tip of the glucose test piece (including the porous material) was impregnated for 1 second in the urine of a healthy person adjusted to a glucose concentration of 1000 mg / 100 ml, and the reflectance (wavelength 600 nm) of the porous material after the impregnation Was measured using a spectrophotometric reflectometer (Murakami Color Research Laboratory, trade name: CMS-35FS). Further, the reflectance was measured in the same manner as Comparative Example 3 except that the gelatin hydrolyzate was not added. The results are shown in Tables 6 and 7 below.

(Mixed solution)
0.1M MES buffer (pH 5.7) 100mL
POD (Toyobo Co., Ltd.) Predetermined amount
GOD (Toyobo Co., Ltd.) Predetermined amount
1-Naphthol-3,6-disulfonic acid Na 1g
4-aminoantipyrine 1g
Alginate Na 30mg
Gelatin hydrolyzate 5g

(Table 6)
POD addition amount (KU / 100mL)
10 8 6 4 2 1
Example 3 16.5 17.0 16.1 16.5 16.3 18.9
Comparative Example 3 14.1 14.4 14.5 16.3 22.6 33.8

(Table 7)
GOD addition amount (KU / 100mL)
20 16 12 8 4 2
Example 3 17.8 17.9 18.2 18.5 21.0 25.1
Comparative Example 3 15.8 17.8 18.1 21.5 23.3 2.95

As shown in Table 6 and Table 7, when the amount of GOD or POD added was changed, the reflectance in Comparative Example 3 varied significantly depending on the amount of enzyme, but according to Example 3, the amount of enzyme was reduced. Also, the fluctuation of the reflectance could be suppressed.

  According to the enzyme stabilization method and the sample analysis tool of the present invention, inactivation of the enzyme can be prevented by allowing the proteolysate to coexist even in a dry state. For this reason, the usage-amount of an enzyme can be reduced and the expiration date of a sample analysis tool can be extended significantly.

Claims (23)

A method for stabilizing a dry enzyme, wherein the enzyme coexists with a protein degradation product. The stabilization method according to claim 1, wherein the protein degradation product is at least one selected from the group consisting of gelatin hydrolyzate, casein hydrolyzate, and soybean hydrolyzate. The stabilization method according to claim 1, wherein the protein degradation product contains a polypeptide having a molecular weight of 500 to 10,000. The stable enzyme according to any one of claims 1 to 3, wherein the enzyme is at least one enzyme selected from the group consisting of glucose oxidase, peroxidase, ascorbate oxidase, hexokinase, diaphorase, pyruvate oxidase and cholesterol oxidase. Method. The stabilization method according to claim 4, wherein the enzyme is a combination of glucose oxidase and peroxidase. The porous material is impregnated with a solution containing the enzyme and a protein degradation product, and the enzyme and the protein degradation product are allowed to coexist in a dry state by drying the porous material. The stabilization method described. The stabilization method according to claim 6, wherein the porous material is a filter paper made of cellulose. The stabilization method according to claim 7, wherein 0.01 to 6 KU of the enzyme is retained per 1 cm ^{3 of the} porous material. The stabilization method according to claim 7 or 8, wherein 5 to 200 mg of the protein degradation product is retained per 1 cm ^{3 of the} porous material. The stabilization method according to any one of claims 1 to 9, wherein an addition ratio of the protein degradation product to the enzyme is in a range of 0.8 to 20,000 mg of the protein degradation product with respect to 1 KU of the enzyme. A sample analysis tool in which an enzyme is held in a porous material, wherein the porous material further holds a protein degradation product. The sample analysis tool according to claim 11, wherein the protein degradation product is at least one selected from the group consisting of gelatin hydrolyzate, casein hydrolyzate, and soybean hydrolyzate. The sample analysis tool according to claim 11, wherein the protein degradation product contains a polypeptide having a molecular weight of 500 to 10,000. The enzyme according to any one of claims 11 to 13, wherein the enzyme is at least one enzyme selected from the group consisting of glucose oxidase, peroxidase, ascorbate oxidase, hexokinase, diaphorase, pyruvate oxidase and cholesterol oxidase. Sample analysis tool. The sample analysis tool according to claim 14, wherein the enzyme is a combination of glucose oxidase and peroxidase. The sample analysis tool according to any one of claims 11 to 15, wherein the porous material is at least one selected from the group consisting of a filter paper, a nonwoven fabric, and a woven fabric. The sample analysis tool according to claim 16, wherein the porous material is a filter paper made of cellulose. The sample analysis tool according to any one of claims 11 to 17, wherein 0.8 to 20,000 mg of the protein degradation product is retained per 1 KU of the enzyme. The sample analysis tool according to any one of claims 11 to 18, wherein 0.01 to 6 KU of the enzyme is held per 1 cm ^{3 of the} porous material. The sample analysis tool according to any one of claims 11 to 19, wherein 5 to 200 mg of the protein degradation product is retained per 1 cm ^{3 of the} porous material. The sample analysis tool according to any one of claims 11 to 20, wherein the analysis object is glucose. Furthermore, the sample analysis tool according to any one of claims 11 to 21, wherein a coloring substrate that develops color by oxidation is held in the porous material. The sample analysis tool according to any one of claims 11 to 22, which is produced by impregnating a porous material with a solution containing the enzyme and a protein degradation product, and drying the porous material.