JP2005278626A - Method for stabilizing enzymatic measuring reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing an enzymatic measuring reagent, by which one reagent system or two reagent system enzymatic measuring reagent containing a ferrocyanide compound to avoid the affection of bilirubin can prevent the deterioration, degradation or degeneration of the a color raw material and a coupler due to the ferrocyanide compound, even when one or both of the ferrocyanide compound and an oxidizable coloring reagent coexist, is stable, when stored for a long period, and can accurately be measured without error. <P>SOLUTION: The method for stabilizing the enzymatic measuring reagent is characterized by containing a chelating agent in the enzymatic measuring reagent using the oxidizable coloring reagent comprising the color raw material and the coupler, an oxidizing enzyme and a peroxidase, and simultaneously containing at least one of the oxidizing enzyme and the first metal ion or a substance containing the same. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a method for stabilizing an enzymatic measurement reagent.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

Measuring biological components contained in biological samples such as blood and urine is very useful in diagnosing diseases. Enzymatic measuring methods have become widespread in clinical tests, and various measuring methods have been developed. ing. As such a method, for example, an oxidase is used to generate hydrogen peroxide, which is an oxidizing substance, directly or indirectly from a biological component, and this is mixed with and contacted with peroxidase and an oxidizable coloring reagent. Measure the hydrogen peroxide produced by optically measuring the dye produced from the oxidizable color reagent by the oxidative condensation reaction, and measure the biological components contained in the biological sample. A method can be mentioned.
For example, for measurement of cholesterol, uric acid, glucose, etc., oxidase such as cholesterol oxidase, uricase, and glucose oxidase is used to generate hydrogen peroxide, and the generated hydrogen peroxide is converted to peroxidase and oxidizable coloring reagent. It is measured by optically measuring the dye produced from an oxidizable coloring reagent by an oxidative condensation reaction, and each biological component can be accurately measured.
Such a measuring method is performed by a one-step method or a two-step method. The former is a one-reagent system in which all of the measuring reagent components are combined, and the latter is a two-component method in which the reagent components are divided into two. Use reagent system.

Conventionally, as an oxidizable coloring reagent used in this method, a chromogen that is mainly a hydrogen donor such as phenol or a derivative thereof or an aniline derivative, and a coupler such as 4-aminoantipyrine as a condensation target thereof A combination of these has been used. However, it is also known that these oxidizable coloring reagents are easily affected by bilirubin in a biological sample (see, for example, Non-Patent Document 1).
Moreover, as a method for avoiding the influence of bilirubin in this biological sample, a method in which a ferrocyanide is contained in a measurement reagent is generally used (see, for example, Non-Patent Document 2).
Clinical Chemistry Vol.8, No.1, pp.63-72, 1980 Clinical Chemistry Vol. 26, No. 2, pp. 227-231, 1980

However, in the case of these conventional methods, if the ferrocyanide and the oxidizable coloring reagent are stored in a coexisting state, the chromogen or coupler in which the ferrocyanide is an oxidizable coloring reagent deteriorates during storage, By decomposing or altering, there is a problem that a negative error is caused in the measured value and accurate measurement cannot be performed.
It is also possible to use a two-reagent-based measurement reagent, in which the ferrocyanide is separately contained in the first reagent (or second reagent), and the chromogen and coupler are separately contained in the second reagent (or first reagent). In this case, since the chromogen and coupler coexist in the second reagent (or the first reagent), the chromogen and coupler are oxidized during storage, and the reagent blank rises. There was a problem.
In addition, when the chromogen that is an oxidizable coloring reagent is separately contained in the first reagent (or the second reagent) and the coupler is contained in the second reagent (or the first reagent) in a two-reagent measuring reagent. However, since either the chromogen or the coupler coexists with the ferrocyanide, there is a problem that the chromogen or the coupler deteriorates due to the effect of the ferrocyanide.
That is, when a ferrocyanide and an oxidizable coloring reagent are allowed to coexist, the chromogen or coupler deteriorates, and there is a problem that accurate measurement cannot be performed.

  Therefore, the subject of the present invention is a one-reagent or two-reagent enzymatic measurement reagent, and in order to avoid the influence of bilirubin, an enzymatic measurement reagent containing ferrocyanide in this enzymatic measurement reagent, Even if one or both of ferrocyanide and oxidizable coloring reagent coexist, deterioration, decomposition or alteration of chromogen and coupler due to ferrocyanide is prevented, and it is stabilized even during long-term storage without error. An object of the present invention is to provide a method for stabilizing an enzymatic measurement reagent capable of performing an accurate measurement.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have added a chelating agent to an oxidizable coloring reagent composed of a chromogen and a coupler, an oxidase and an enzymatic measurement reagent using peroxidase. Thus, it has been found that the enzymatic measurement reagent can be stabilized without deteriorating the oxidizable color reagent even when either or both of the oxidizable color reagent and the ferrocyanide coexist. The invention has been completed.

That is, the present invention provides the following inventions.
(1) An oxidizable coloring reagent comprising a chromogen and a coupler, and an enzymatic measurement reagent using an oxidase and a peroxidase, wherein at least one of the oxidizable coloring reagent and the first metal ion or this A method for stabilizing an enzymatic measurement reagent, comprising the step of containing a chelating agent in a measurement reagent simultaneously containing a contained substance.

(2) The method for stabilizing an enzymatic measurement reagent according to (1), wherein the coupler is 4-aminoantipyrine.

(3) The first metal ion or a substance containing the first metal ion degrades, decomposes or alters the reagent component contained in the enzymatic measurement reagent, or causes an error in the measurement value. The method for stabilizing an enzymatic measurement reagent according to (1) or (2) above.

(4) The method for stabilizing an enzymatic measurement reagent according to any one of (1) to (3), wherein the first metal ion or a substance containing the first metal ion is a ferrocyanide.

(5) The method for stabilizing an enzymatic measurement reagent according to any one of (1) to (4), wherein the chelating agent coordinates to the first metal ion or a substance containing the first metal ion.

(6) The chelating agent is gluconic acid, ethylenediaminetetraacetic acid, ethylenediaminedipropionic acid, bis (aminophenyl) ethylene glycol tetraacetic acid, citric acid, succinic acid, tartaric acid, N- [tris (hydroxymethyl) methyl] glycine, At least one substance selected from the group consisting of N, N-bis (2-hydroxyethyl) glycine, salicylic acid, N- (2-acetamido) iminodiacetic acid, oxalic acid, thiosulfuric acid, thiocyanic acid or a salt thereof The method for stabilizing an enzymatic measurement reagent according to any one of (1) to (5).

(7) The method for stabilizing an enzymatic measurement reagent according to any one of (1) to (6), further including a second metal ion or a substance containing the second metal ion.

(8) The enzymatic measurement reagent according to (7), wherein the second metal ion or a substance containing the second metal ion stabilizes and / or activates a reagent component contained in the enzymatic measurement reagent. Stabilization method.

(9) The reagent component stabilized and / or activated by the second metal ion or a substance containing the second metal ion is cholesterol esterase, lipoprotein lipase or phospholipase, as described in (7) or (8) above. Stabilization method for enzymatic measurement reagents.

(10) The method for stabilizing an enzymatic measurement reagent according to any one of (7) to (9), wherein the second metal ion is calcium ion or magnesium ion.

(11) The (7), wherein the chelating agent coordinates to the first metal ion or a substance containing the same but does not coordinate to the second metal ion or the substance containing the second metal ion. The stabilization method of the enzymatic measurement reagent in any one of-(10).

(12) The chelating agent is at least selected from the group consisting of gluconic acid, succinic acid, tartaric acid, N- [tris (hydroxymethyl) methyl] glycine, N, N-bis (2-hydroxyethyl), or salicylic acid. The method for stabilizing an enzymatic measurement reagent according to (11), which is one substance.

  In the method for stabilizing an enzymatic measurement reagent of the present invention, by adding a chelating agent to an oxidizable coloring reagent comprising a chromogen (hydrogen donor) and a coupler, and an enzymatic measurement reagent using an oxidase and peroxidase, A one-reagent or two-reagent enzymatic measurement reagent that contains ferrocyanide in the enzymatic measurement reagent to avoid the effects of bilirubin. Ferrocyanide and oxidizable color development Even when one or both of the reagents coexist, it is possible to prevent deterioration, decomposition or alteration of the chromogen and coupler due to ferrocyanide, stabilize during long-term storage, and perform accurate measurement without error. It became so.

(1) Oxidizable coloring reagent The oxidizable coloring reagent used in the present invention is a peroxidase that acts as a peroxidase when measuring biological components by measuring hydrogen peroxide produced by the action of oxidase. A reagent composed of a combination of a chromogen and a coupler for producing a dye by oxidative condensation with hydrogen.

  A chromogen is a hydrogen donor that releases hydrogen during the oxidative condensation reaction, and is oxidized by the action of peroxidase and oxidatively condensed with a coupler such as 4-aminoantipyrine described later to produce a dye. If it does not specifically limit, For example, a phenol or its derivative (s), or an aniline derivative etc. can be mentioned.

  Here, examples of the phenol derivative include 4-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, and salts thereof. .

  Examples of aniline derivatives include N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), and N-ethyl. -N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N- ( 2-carboxyethyl) -N-ethyl-3,5-dimethoxyaniline (CEDB), N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N-ethyl-N- (2-hydroxy-3-sulfo) Propyl) aniline (ALOS), N-ethyl-N- (3-sulfopropyl) aniline (ALPS), N- (3-sulfopropyl) aniline (HALPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3-methoxyaniline (TOOS), N- (2-carboxyethyl) -N-ethyl-3-methylaniline (CEMB), or N (2-carboxyethyl) -N- ethyl-3-methoxyaniline (Cemo), or may be their salts, and the like.

  The concentration of the chromogen is not particularly limited, but is preferably in the range of 0.05 to 20 mM, preferably in the range of 0.1 to 5 mM in the measurement reaction solution after mixing the sample and the measurement reagent. It is particularly preferred.

The coupler is not particularly limited as long as it is oxidized by the action of peroxidase and oxidatively condensed with the chromogen to produce a dye. For example, 4-aminoantipyrine or a derivative thereof, phenylenediamine, 3-methyl- And 2-benzothiazolinone hydrazone (MBTH).
In the present invention, the coupler is preferably 4-aminoantipyrine.
Further, the concentration of the coupler is not particularly limited, but is preferably in the range of 0.05 to 10 mM, particularly preferably in the range of 0.1 to 5 mM in the measurement reaction solution after mixing the sample and the measurement reagent. .

(2) Oxidase In the present invention, an oxidase refers to a substance that catalyzes a reaction for generating hydrogen peroxide, which is an oxidizing substance, directly or indirectly from a biological component.
Here, examples of the oxidase include cholesterol oxidase, uricase, glucose oxidase, glycerol oxidase, xanthine oxidase, and pyruvate oxidase.
In the present invention, the concentration of the oxidase is not particularly limited, but is preferably in the range of 7 to 800 units / L in the measurement reaction solution after mixing the sample and the measurement reagent.

(3) Peroxidase In the present invention, the peroxidase may be of any origin, such as those derived from animals such as humans or cows, those derived from plants such as horseradish, or microorganisms such as bacteria or molds. The thing etc. can be mentioned.
Examples of the peroxidase include those prepared by a gene recombination technique in which a peroxidase gene such as a microorganism is incorporated into a microorganism such as Escherichia coli, or peroxidase prepared from a microorganism whose properties have been improved by gene modification or the like.
The concentration of peroxidase is not particularly limited, but is preferably 20 units / L or more in the measurement reaction liquid after mixing the sample and the measurement reagent.

(4) First metal ion or substance containing the same In the present invention, the enzymatic measurement reagent comprises at least one of the oxidizable coloring reagent and the first metal ion or a substance containing the first metal ion. In the same reagent as the first reagent or the second reagent.
The first metal ion or a substance containing the first metal ion degrades, decomposes or alters reagent components such as the oxidizable coloring reagent contained in the enzymatic measurement reagent, or has an error in the measurement value. It will cause.
Here, examples of the first metal ion include divalent or trivalent iron ions.
Moreover, as a substance containing the iron ion as a 1st metal ion, a ferrocyanide etc. can be mentioned, for example. Examples of the ferrocyanide include potassium ferrocyanide and sodium ferrocyanide.

(5) Chelating agent The present invention is characterized in that a chelating agent is contained in the enzymatic measurement reagent. In the present invention, by containing this chelating agent in the enzymatic measurement reagent, even when either or both of the oxidizable color reagent and the ferrocyanide coexist, the oxidizable color reagent is converted to the ferrocyanide. Thus, accurate measurement can be performed without deterioration.

  Here, the chelating agent needs to coordinate to the first metal ion or a substance containing the first metal ion. Examples of such chelating agents include gluconic acid, ethylenediaminetetraacetic acid [EDTA], ethylenediaminenipropionic acid [EDDP], bis (aminophenyl) ethylene glycol tetraacetic acid [BAPTA], citric acid, succinic acid, tartaric acid, N -[Tris (hydroxymethyl) methyl] glycine [tricine], N, N-bis (2-hydroxyethyl) glycine [bicine], salicylic acid, N- (2-acetamido) iminodiacetic acid [ADA], oxalic acid, thio Examples thereof include sulfuric acid, thiocyanic acid or a salt thereof.

  The concentration of the chelating agent is not particularly limited as long as it can coordinate with all of the first metal ions contained in the enzymatic measurement reagent, but in the measurement reaction liquid after mixing the sample and the measurement reagent. 0.075 to 200 mM is preferable, and it is particularly preferable that the range is 0.5 to 50 mM.

In addition, the method for stabilizing an enzymatic measurement reagent of the present invention is a known one-reagent or two-reagent enzymatic measurement reagent using an oxidase, for example, directly or indirectly by acting an oxidase on a biological component. Hydrogen peroxide is generated, and the obtained hydrogen peroxide is mixed with a peroxidase and an oxidizable color reagent, and a conventionally known enzymatic measurement reagent for measuring the generated color product is added to at least one of the aforementioned chelating agents. It can be carried out by containing one kind.
Furthermore, the present invention can be implemented in any enzymatic measurement reagent that contains either one or both of the oxidizable coloring reagent and the first metal ion or a substance containing the same at the same time.
In addition, this chelating agent may use only 1 type, or may use multiple types simultaneously.

(6) Second metal ion or substance containing the same In the enzymatic measurement reagent of the present invention, the second metal ion or the substance different from the first metal ion or the substance containing the first metal ion is contained. The substance to do may be included.
As this 2nd metal ion or a substance containing this, what stabilizes and / or activates the reagent component contained in an enzymatic measurement reagent can be mentioned, for example.
More specifically, examples of these second metal ions include calcium ions and magnesium ions.
In addition to the reagent components described above, the present invention is suitable for an enzymatic measurement reagent containing a second metal ion or a substance containing the second metal ion.

  That is, the present inventors have found that iron ions in ferrocyanide are involved in the degradation of chromogens and couplers that are oxidizable coloring reagents, and coordinated a chelating agent to iron ions in ferrocyanide. It was found that the deterioration of the chromogen and the coupler can be suppressed by making it.

  However, in the case of an enzymatic measurement reagent that uses an enzyme that is stabilized and / or activated by calcium ions (second metal ions), such as microorganism-derived cholesterol esterase, calcium is contained in the enzymatic measurement reagent. It is necessary to contain ions. In this case, the chelating agent added for the purpose of coordinating the iron ions in the ferrocyanide also coordinates the calcium ions, destabilizing the cholesterol esterase.

  In such a case, the ferrocyanide can be oxidized by selecting a chelating agent that coordinates to the iron ion in the ferrocyanide but not (or difficult to coordinate) to the calcium ion. It is possible to suppress deterioration of the coloring reagent and not to affect the stabilization of reagent components such as enzymes.

  That is, by selecting a chelating agent that coordinates to the first metal ion or a substance containing the first metal ion but does not coordinate to the second metal ion or the substance containing the first metal ion, It is possible to suppress deterioration of the oxidizable coloring reagent and to prevent the stabilization and / or activation of the reagent component contained in the enzymatic measurement reagent by the second metal ion.

  Here, as a chelating agent that coordinates to the first metal ion or a substance containing the same but does not coordinate to the second metal ion or the substance containing the same, for example, gluconic acid, succinic acid, Examples thereof include tartaric acid, N- [tris (hydroxymethyl) methyl] glycine [tricine], N, N-bis (2-hydroxyethyl) glycine [bicine], and salicylic acid.

(7) Reagent component stabilized and / or activated by the second metal ion or a substance containing the second ion or stabilized and / or activated by the substance containing the second metal ion in the present invention Examples of the reagent component that is stabilized and / or activated by calcium ions or substances containing the same include cholesterol esterase and phospholipase.
Examples of reagent components that are stabilized and / or activated by magnesium ions or substances containing the same include, for example, lipoprotein lipase.
In the present invention, the reagent component that is stabilized and / or activated by the second metal ion or a substance containing the second metal ion is preferably cholesterol esterase.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by this Example.

[Example 1]
(Confirmation of stabilizing effect of enzymatic assay reagent according to the present invention-1)
In the present invention, the stabilizing effect of the enzymatic measurement reagent when sodium gluconate, which is a chelating agent, is contained in the enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of reagent for enzymatic measurement (1) Preparation of first reagent The following reagent components are dissolved in pure water so as to have the stated concentrations, and the pH is adjusted to 5.4 and 6.3 (20 ° C), respectively. Then, six kinds of first reagents having different pHs and concentrations of sodium gluconate as a chelating agent were prepared.
Good buffer solution (Saikyo Kasei) 100 mM
4-Aminoantipyrine (Daiichi Kagaku) 0.11 g / L
Potassium ferrocyanide (domestic chemistry) 31μM
Calcium chloride (domestic chemistry) 2 mM
Cholesterol esterase (Toyobo) 500 units / L
Sodium gluconate (Tokyo Kasei) 23 mM, 46 mM, or no addition (0 mM)

(2) Preparation of Second Reagent The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 6.9 (20 ° C.) to prepare a second reagent.
Good buffer (Saikyo Kasei) 20mM
N- (2-hydroxy-3-sulfopropyl)
-3,5-dimethoxyaniline [HDAOS] (Saikyo Kasei) 2 mM
Peroxidase (Toyobo) 8000 units / L
Cholesterol oxidase (Amano Pharmaceutical) 1320 units / L

2. Storage of the first reagent and the second reagent The six types of first reagent prepared in (1) above and the second reagent prepared in (2) above are stored at 10 ° C. and 25 ° C. for 196 days, respectively. did.

3. Measurement of 4-aminoantipyrine content The 4-aminoantipyrine content of each of the six types of first reagents was measured at the start of storage, after 147 days of storage, and after 196 days.
The content of 4-aminoantipyrine was measured by measuring the absorbance at a wavelength of 505 nm.
Then, the absorbance after 147 days and 196 days after storage (4-aminoantipyrine content) is divided by the absorbance at the start of storage (4-aminoantipyrine content) and multiplied by 100 to obtain 147 days and 196 days after storage. The residual ratio (%) of 4-aminoantipyrine was calculated.

4). Measurement Results Table 1 shows the measurement results of the content of 4-aminoantipyrine.
In this table, the numerical value in parenthesis indicates the residual ratio (%) of 4-aminoantipyrine after storage calculated as described above.

  As is clear from Table 1, the content (absorbance) of 4-aminoantipyrine is reduced by storing the first reagent in the case where the first reagent does not contain sodium gluconate (0 mM). I understand.

  That is, the pH adjusted to 5.4 is 3.7% after 147 days of storage at 10 ° C. and 2.0% after 196 days when the absorbance of 4-aminoantipyrine at the start of storage is 100%. It turns out that it has fallen to. Further, it can be seen that after 147 days of storage at 25 ° C., it decreased to 3.3%, and after 196 days, it decreased to 4.5%. The pH adjusted to 6.3 was 44.2% after 147 days when stored at 10 ° C. and 13.3 after 196 days when the absorbance of 4-aminoantipyrine at the start of storage was 100%. It turns out that it has fallen to%. Furthermore, it can be seen that the storage at 25 ° C. decreased to 3.5% after 147 days and 196 days.

  On the other hand, when sodium gluconate, which is a chelating agent, is contained in the first reagent, the one adjusted to pH 5.4 is the residual 4-aminoantipyrine when the sodium gluconate concentration is 23 mM and 46 mM. The rates were 61.0% and 74.9% after 147 days of storage at 10 ° C., and 53.9% and 73.0% after 196 days, respectively. Further, after 147 days of storage at 25 ° C., they are 18.9% and 48.6%, respectively, and after 196 days, they are 5.0% and 28.8%, respectively.

  In addition, when adjusted to pH 6.3, the residual ratio of 4-aminoantipyrine when the sodium gluconate concentration was 23 mM and 46 mM was 96.3% and 101.4% after 147 days of storage at 10 ° C. After 196 days, they were 97.6% and 105.8%, respectively. Further, after 147 days of storage at 25 ° C., they are 85.2% and 97.9%, respectively, and after 196 days, they are 83.4% and 101.6%, respectively.

From these results, it is understood that the residual ratio of 4-aminoantipyrine is significantly improved by the presence of sodium gluconate, which is a chelating agent, in the enzymatic measurement reagent in which potassium ferrocyanide and 4-aminoantipyrine coexist. . It can also be seen that the residual ratio of 4-aminoantipyrine is improved as the concentration of sodium gluconate is increased.
From the above, it was confirmed that 4-aminoantipyrine can be stabilized by including sodium gluconate as a chelating agent in an enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist.

[Example 2]
(Confirmation of stabilizing effect of enzymatic measuring reagent according to the present invention-2)
In the present invention, the stabilizing effect of the enzymatic measurement reagent when the chelating agent is contained in the enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of enzymatic measurement reagent (1) Preparation of first reagent Each of the eight chelating agents described in Table 2 contains (or does not contain) at a concentration of 3 mM, 15 mM, or 30 mM, and pH is 6. Except for adjusting to 9, 25 kinds of first reagents were prepared in the same manner as in the first reagent (1) of Example 1 (1).

(2) Preparation of Second Reagent A second reagent was prepared in the same manner as the second reagent of Example 1, 1 (2).

2. Storage of First Reagent and Second Reagent The 25 types of first reagent prepared in (1) above and the second reagent prepared in (2) above were each stored at 37 ° C. for 36 days.

3. Measurement of 4-aminoantipyrine content The content of 4-aminoantipyrine of each of the 25 types of first reagents was measured in the same manner as in Example 1 at the start of storage and after 7 days, 19 days, and 36 days after storage. It was measured.

4). Measurement Results Table 2 shows the measurement results of the content of 4-aminoantipyrine.
In this table, the numerical value in parenthesis indicates the residual ratio (%) of 4-aminoantipyrine after storage calculated in the same manner as described in Example 1-3.

  As is apparent from Table 2, it can be seen that the content (absorbance) of 4-aminoantipyrine decreases with the passage of the storage days when the first reagent does not contain a chelating agent (0 mM).

  That is, assuming that the absorbance of 4-aminoantipyrine at the start of storage is 100%, it has decreased to 80.7% after 7 days, 48.5% after 19 days, and 14.8% after 36 days. I understand that.

  On the other hand, when sodium gluconate, citric acid, or tartaric acid is contained in the first reagent, the residual ratio of 4-aminoantipyrine is 94% or more after 7 days of storage, 84% or more after 19 days, Even after 36 days, it is 71% or more. In addition, it can be seen that the remaining rate of 4-aminoantipyrine is higher for other chelating agents as well, to some extent, as compared with the case where no chelating agent is contained.

From these results, it can be seen that the residual ratio of 4-aminoantipyrine is improved by adding a chelating agent to the reagent in which potassium ferrocyanide and 4-aminoantipyrine coexist.
From the above, it was confirmed that 4-aminoantipyrine can be stabilized by adding a chelating agent to a reagent in which ferrocyanide and 4-aminoantipyrine coexist.

Example 3
(Confirmation of stabilizing effect of enzymatic measurement reagent according to the present invention-3)
In the present invention, the stabilizing effect of the enzymatic measurement reagent when the chelating agent is contained in the enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of enzymatic measurement reagent (1) Preparation of first reagent Each of the 10 chelating agents listed in Table 3 is contained (or not contained) at the concentrations shown in the table, and the pH is adjusted to 6.5. Except that, 30 types of first reagents were prepared in the same manner as the first reagent of Example 1, (1) (1).

(2) Preparation of Second Reagent A second reagent was prepared in the same manner as the second reagent of Example 1, 1 (2).

2. Storage of First Reagent and Second Reagent The 30 first reagents prepared in (1) above and the second reagent prepared in (2) above were stored at 37 ° C. for 36 days.

3. Measurement of 4-aminoantipyrine content The content (absorbance) of 4-aminoantipyrine of each of the 30 kinds of first reagents was the same as in Example 1 at the start of storage and after 7 days, 19 days, and 36 days. It measured by the method of.

4). Measurement Results Table 3 shows the measurement results of the content of 4-aminoantipyrine.
In this table, the numerical value in parenthesis indicates the residual ratio (%) of 4-aminoantipyrine after storage calculated in the same manner as described in Example 1-3.

  As is clear from Table 3, it can be seen that the content (absorbance) of 4-aminoantipyrine decreases with the passage of the storage days when the first reagent does not contain a chelating agent (0 mM).

  That is, assuming that the content (absorbance) of 4-aminoantipyrine at the start of storage is 100%, 79.5% after storage, 9.0% after 19 days, and -5.0% after 36 days It turns out that it has fallen to.

  In contrast, when sodium gluconate, disodium EDTA, citric acid, tartaric acid, or salicylic acid is contained in the first reagent, the residual ratio of 4-aminoantipyrine is 88% or more after 7 days of storage. Even after 19 days, it is over 62%. In addition, it can be seen that the remaining rate of 4-aminoantipyrine is higher for other chelating agents as well, to some extent, as compared with the case where no chelating agent is contained.

From these results, it can be seen that the residual ratio of 4-aminoantipyrine is improved by adding a chelating agent to the enzymatic measurement reagent in which potassium ferrocyanide and 4-aminoantipyrine coexist.
From the above, it was confirmed that 4-aminoantipyrine can be stabilized by adding a chelating agent to an enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist.

Example 4
(Confirmation of stabilizing effect of enzymatic measuring reagent according to the present invention-4)
In the present invention, the stabilizing effect of the enzymatic measurement reagent when the chelating agent is contained in the enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of Enzymatic Measurement Reagent (1) Preparation of First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, pH is adjusted to 7.0 (20 ° C.), Prepared.
Good buffer (Saikyo Kasei) 20mM
N- (2-carboxyethyl) -N-ethyl
-3-Methylaniline [CEMB] 1.5 mM
Glycerol kinase 150 units / L
Glycerol-3-phosphate oxidase 3,000 units / L
Adenosine triphosphate disodium 0.5 mM

(2) Preparation of Second Reagent Dissolve each of the 11 types of chelating agents listed in Table 4 in pure water at the concentrations indicated in the table and the following reagent components to the indicated concentrations, and adjust the pH to 6 Each was adjusted to 0.0 (20 ° C.) to prepare 23 types of second reagents.
Good buffer solution (Saikyo Kasei) 50 mM
4-Aminoantipyrine (Daiichi Kagaku) 0.457 g / L
Potassium ferrocyanide (domestic chemistry) 30μM
Peroxidase (Toyobo) 600 units / L
Lipoprotein lipase (Asahi Kasei) 100,000 units / L

2. Storage of First Reagent and Second Reagent The first reagent prepared in (1) above and the 23 second reagents prepared in (2) above were stored at 25 ° C. for 28 days.

3. Measurement of 4-Aminoantipyrine Content The 4-aminoantipyrine content (absorbance) of each of the 23 types of second reagents was measured at the start of storage and after 28 days of storage.
The content of 4-aminoantipyrine was measured by measuring the absorbance at a wavelength of 585 nm.
Then, the absorbance after 28 days of storage (4-aminoantipyrine content) is divided by the absorbance at the start of storage (4-aminoantipyrine content) and multiplied by 100, thereby remaining 4-aminoantipyrine after 28 days of storage. The rate (%) was calculated.

4). Measurement Results Table 4 shows the measurement results of the content of 4-aminoantipyrine.
In this table, the numerical value in parenthesis indicates the residual ratio (%) of 4-aminoantipyrine after storage calculated in the same manner as described in Example 1-3.

As is clear from Table 4, it can be seen that the content (absorbance) of 4-aminoantipyrine is reduced in the case where the chelating agent is not contained in the second reagent (0 mM).
That is, it can be seen that the 4-aminoantipyrine content (absorbance) at the start of storage is reduced to 15.5% after 28 days when the content is 100%.

  On the other hand, when sodium gluconate, disodium EDTA, citric acid, salicylic acid, or sodium thiosulfate is contained in the second reagent, the residual ratio of 4-aminoantipyrine varies depending on the concentration. More than 56% later. In addition, it can be seen that the remaining rate of 4-aminoantipyrine is higher for other chelating agents as well, to some extent, as compared with the case where no chelating agent is contained.

From these results, it can be seen that the residual ratio of 4-aminoantipyrine is improved by adding a chelating agent to the enzymatic measurement reagent in which potassium ferrocyanide and 4-aminoantipyrine coexist.
From the above, it was confirmed that 4-aminoantipyrine can be stabilized by adding a chelating agent to an enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist.

Example 5
(Confirmation of influence on cholesterol esterase activity by the present invention-1)
In the present invention, the influence on the cholesterol esterase in the reagent when the chelating agent is contained in the enzymatic measurement reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of enzymatic measurement reagent (1) Preparation of first reagent Each of the nine chelating agents listed in Table 5 was contained (or not contained) at the concentrations listed in the table, and the pH was 6.9 (20 The first reagent of 28 types was prepared in the same manner as the first reagent of (1) of Example 1 except that the temperature was adjusted to (° C.).

(2) Preparation of Second Reagent The second reagent prepared in (1) of Example 1 was used as it was.

2. Storage of first reagent and second reagent The 28 types of first reagent prepared in (1) above and the second reagent prepared in (2) above were stored at 10 ° C. for 36 days.

3. Measurement of Cholesterol Esterase Activity Values The cholesterol esterase activity values (absorbance) of each of the 28 types of first reagents were measured at the start of storage, after 7 days of storage, and after 36 days.
The cholesterol esterase activity value (absorbance) was measured according to the cholesterol esterase activity measuring method described on page 54 of the catalog “TOYOBO ENZYMES” (issued July 1, 2002).
Then, the absorbance after 7 days and 36 days after storage (cholesterol esterase activity value) is divided by the absorbance at the start of storage of the first reagent without addition of a chelating agent (cholesterol esterase activity value) and multiplied by 100. The residual ratio (%) of cholesterol esterase activity after day and day 36 was calculated.

4). Measurement Results Table 5 shows the measurement results of the cholesterol esterase activity value.
In this table, the numerical value in parenthesis indicates the residual rate (%) of cholesterol esterase activity after storage calculated as described above.

  As can be seen from Table 5, when EDTA disodium is contained in the first reagent, the activity value (absorbance) of cholesterol esterase is decreased from the beginning of storage of the reagent (day 0). .

That is, when the EDTA disodium concentration is 3 mM, 15 mM, or 30 mM, the residual rate of cholesterol esterase activity is 67. 5 at the start of storage when the activity value (absorbance) of cholesterol esterase at the start of storage is 100%. 5%, 31.6%, and 27.0%. After 7 days, they were 45.1%, 18.8%, and 15.5%, respectively, and after 36 days, 26.1% and 9.5, respectively. %, 7.9%.
In addition, when citric acid or ADA is contained in the first reagent, there is a difference depending on the concentration and the number of storage days, but the cholesterol esterase activity value (absorbance) at the start of storage is assumed to be at least 70%. It turns out that it has fallen to.

  On the other hand, when sodium gluconate, tricine, bicine, succinic acid, tartaric acid, or salicylic acid is contained in the first reagent, the residual rate of cholesterol esterase activity varies depending on the concentration and the number of storage days. Almost no decline.

  From these results, disodium EDTA, citric acid, or ADA decreases the activity of cholesterol esterase, but sodium gluconate, tricine, bicine, succinic acid, tartaric acid, or salicylic acid adversely affects the activity of cholesterol esterase. I understand that it is not.

  From the above results and the results of Examples 1 to 4, it coordinates to iron ions (first metal ions) in ferrocyanide, but does not coordinate to calcium ions (second metal ions) ( By selecting a chelating agent that is difficult to coordinate), ferrocyanide suppresses degradation of 4-aminoantipyrine (oxidizing chromogenic reagent) and cholesterol esterase (enzymatic measurement) by a second metal ion It was confirmed that there was no adverse effect on the stabilization of the reagent component contained in the reagent.

Example 6
(Confirmation of influence on cholesterol esterase activity by the present invention-2)
In the present invention, the effect on the cholesterol esterase in the reagent when the chelating agent is contained in a reagent in which ferrocyanide and 4-aminoantipyrine coexist was confirmed.

1. Preparation of enzymatic measurement reagent (1) Preparation of first reagent Each of 11 types of chelating agents shown in Table 6 is contained (or not) at the concentrations shown in the table, and pH is 6.5 (20 ° C. Except for the adjustment to (1), 33 kinds of first reagents were prepared in the same manner as the first reagent (1) of Example 1 (1).

(2) Preparation of Second Reagent The second reagent prepared in (1) of Example 1 was used as it was.

2. Storage of First Reagent and Second Reagent The 33 types of first reagent prepared in (1) above and the second reagent prepared in (2) above were stored at 10 ° C. for 36 days.

3. Measurement of cholesterol esterase activity value The cholesterol esterase activity value (absorbance) of each of the 33 kinds of first reagents was measured in the same manner as in Example 5 at the start of storage, 7 days after storage, and 36 days after storage.

4). Measurement Results Table 6 shows the measurement results of cholesterol esterase activity values.
In this table, the numerical values in parentheses indicate the residual rate (%) of cholesterol esterase activity after storage calculated in the same manner as described in Example 5-3.

  As is clear from Table 6, when EDTA disodium, citric acid, ADA, EDDP, or BAPTA was contained in the first reagent, the activity value of cholesterol esterase (from the start of storage of the reagent (day 0) ( It can be seen that the absorbance is decreased.

  That is, when the first reagent contains disodium EDTA, citric acid, ADA, EDDP, or BAPTA, the residual rate of cholesterol esterase activity is greatly reduced when the concentration of the chelating agent is high. I understand.

  On the other hand, when sodium gluconate, tricine, bicine, succinic acid, tartaric acid, or salicylic acid is contained in the first reagent, the residual rate of cholesterol esterase activity varies depending on the concentration and the number of storage days. Almost no decline.

  From these results, disodium EDTA, citric acid, ADA, EDDP, or BAPTA decreases the activity of cholesterol esterase, but sodium gluconate, tricine, bicine, succinic acid, tartaric acid, or salicylic acid has an activity of cholesterol esterase. It can be seen that there is no adverse effect on

  From the above results and the results of Examples 1 to 4, it coordinates to iron ions (first metal ions) in ferrocyanide, but does not coordinate to calcium ions (second metal ions) ( By selecting a chelating agent that is difficult to coordinate), ferrocyanide suppresses degradation of 4-aminoantipyrine (oxidizing chromogenic reagent) and cholesterol esterase (enzymatic measurement) by a second metal ion It was confirmed that there was no adverse effect on the stabilization of the reagent component contained in the reagent.

Claims (12)

  An oxidizable coloring reagent comprising a chromogen and a coupler, and an enzymatic measurement reagent using an oxidase and a peroxidase, and at least one of the oxidizable coloring reagent and a first metal ion or a substance containing the same A method for stabilizing an enzymatic measurement reagent, characterized in that a chelating agent is contained in a measurement reagent simultaneously containing   The method for stabilizing an enzymatic measurement reagent according to claim 1, wherein the coupler is 4-aminoantipyrine.   The first metal ion or a substance containing the same degrades, decomposes or alters reagent components contained in the enzymatic measurement reagent, or causes an error in a measurement value. The method for stabilizing an enzymatic measurement reagent according to claim 1 or 2.   The method for stabilizing an enzymatic measurement reagent according to any one of claims 1 to 3, wherein the first metal ion or a substance containing the first metal ion is a ferrocyanide.   The method for stabilizing an enzymatic measurement reagent according to any one of claims 1 to 4, wherein the chelating agent coordinates to the first metal ion or a substance containing the first metal ion.   The chelating agent is gluconic acid, ethylenediaminetetraacetic acid, ethylenediaminenipropionic acid, bis (aminophenyl) ethylene glycol tetraacetic acid, citric acid, succinic acid, tartaric acid, N- [tris (hydroxymethyl) methyl] glycine, N, N -At least one substance selected from the group consisting of bis (2-hydroxyethyl) glycine, salicylic acid, N- (2-acetamido) iminodiacetic acid, oxalic acid, thiosulfuric acid, thiocyanic acid or a salt thereof The method for stabilizing an enzymatic measurement reagent according to any one of claims 1 to 5.   The method for stabilizing an enzymatic measurement reagent according to any one of claims 1 to 6, further comprising a second metal ion or a substance containing the second metal ion in the enzymatic measurement reagent.   The method for stabilizing an enzymatic measurement reagent according to claim 7, wherein the second metal ion or a substance containing the second metal ion stabilizes and / or activates a reagent component contained in the enzymatic measurement reagent.   The reagent component that is stabilized and / or activated by the second metal ion or a substance containing the second metal ion is at least one reagent component selected from the group consisting of cholesterol esterase, lipoprotein lipase, or phospholipase. Item 9. A method for stabilizing an enzymatic measurement reagent according to Item 7 or Item 8.   The method for stabilizing an enzymatic measurement reagent according to any one of claims 7 to 9, wherein the second metal ion is calcium ion and / or magnesium ion.   The chelating agent coordinates to the first metal ion or a substance containing the same, but does not coordinate to the second metal ion or the substance containing the same. The method for stabilizing an enzymatic measurement reagent according to any one of the above.   The chelating agent is at least one selected from the group consisting of gluconic acid, succinic acid, tartaric acid, N- [tris (hydroxymethyl) methyl] glycine, N, N-bis (2-hydroxyethyl) glycine, or salicylic acid. The method for stabilizing an enzymatic measurement reagent according to claim 11, which is a substance.