JP2000228997A - Reagent composition for measuring electrolyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a reagent composition for measuring electrolytes such as calcium ions and chlorine ones in body fluid, or the like by formulating an inactivation-type α-amylase capable of reversibly being reactivated with an electrolyte and a chelating agent separately. SOLUTION: When this α-amylase-using reagent composition for measuring electrolytes consists of two reagents, the reagent composition is prepared by formulating an inactivation-type α-amylase capable of reversibly being reactivated with an electrolyte and a chelating agent separately. The first reagent is prepared by formulating a substrate for an α-amylase (e.g. 2-chloro-4- nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside or the like), a chelating agent [e.g. 1,2-bis(o-aminophenoxy)ethanetetraacetic acid or the like] and an α-amylase inhibiting substance (e.g. N-ethylmaleimide or the like). The second reagent is prepared by formulating an inactivation-type α-amylase capable of reversibly being reactivated with an electrolyte. Thus, the objective reagent composition for measuring electrolytes in blood and urine is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent composition for measuring electrolytes in body fluids, especially blood or urine, for example, electrolytes such as calcium ions and chloride ions. More specifically, the present invention relates to a reagent composition for measuring an electrolyte utilizing α-amylase.

[0002]

2. Description of the Related Art Since the concentration of electrolytes in a living body is usually strictly regulated by metabolism, measurement of electrolytes in body fluids is the most common analysis in biochemical clinical tests as a barometer of biological functions. Yes, and by measuring these, various diseases are diagnosed. For example, measurement of the amount of calcium ions in serum is performed by measuring hypocalcemia as diseases such as hypoproteinemia, hypophosphatemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism, rickets, and high calcium. The blood is used for diagnosis of diseases such as bone tumor, Addison's disease, emphysema, hyperparathyroidism, and renal failure.
Further, for example, measurement of the amount of chloride ion in serum can be performed as hypochloremia, such as hypotonic dehydration, hyperglucocorticoid disease, diseases such as respiratory acidosis, and hyperchloremia as hypertonic dehydration, urine. It is used to diagnose diseases such as tubular acidosis and respiratory alkalosis.

Conventionally, methods for measuring calcium ions in body fluids include (1) titration, (2) colorimetry, (3) atomic absorption, (4) flame photometry, and (5) electrode. , (6)
Enzymatic methods are known.

[0004] The titration method (1) is a method of chemically titrating with oxalate or chelate. However, this method is complicated, and a large amount of sample may be obtained due to differences in measured values by the practitioner. Cannot be processed in a short time.
In the colorimetric method (2), o-CP is used as the color former.
The method using C (orthocresol phthalein complexon) is a method applicable to a general-purpose automatic analyzer, but has a drawback that the absorbance changes depending on temperature and time and is affected by magnesium ions. .

Further, the atomic absorption method (3) has drawbacks such as the necessity of diluting the sample and the skill of the technique. The flame photometer method (4) has problems in specificity and reproducibility. Further, the electrode method of the above (5)
It has drawbacks such as being affected by pH, and complicated to maintain the device in a constant state.

The enzymatic method (6) described above includes a method using (a) phospholipase D (JP-A-62-19552).
No. 97), (b) a method using calmodulin (JP-A-62-36199), and (c) a method using α-amylase (JP-B-6-87798).

The above method (a) has a drawback that it is difficult to uniformly prepare a substrate, and the reaction takes a long time. In the method (b), the sample is diluted (100 to 1).
000 times). Also,
The method (c) is based on the inactivation type α-
The amylase is reactivated, the sugar substrate is decomposed, and the decomposed product is measured.By measuring the calcium ion in the body fluid, the inactivated α-amylase is reactivated by the calcium ion, and the sugar substrate is converted. This is a method for measuring calcium ions in a body fluid by decomposing and measuring the decomposed product, which solves the problems in the above methods (1) to (5), and also (6) (a), It is actually used in the field of clinical testing as an enzyme method that is more accurate and simpler than the method (b).

On the other hand, as a method for measuring chloride ions in body fluids, there have been conventionally used (a) a coulometric titration method, (b) an ion electrode method,
(C) Colorimetric method, (d) Enzymatic method and the like are known. The coulometric titration method of (a) and the ion electrode method of (b) have problems such as the necessity of a dedicated device, the necessity of careful maintenance and management of the device, and the poor sample analysis efficiency. Also,
Although the colorimetric method (c) includes a method using mercury thiocyanate and iron nitrate, it has a drawback that a special treatment is required because a waste liquid containing cyan and mercury is generated after the measurement.

In the enzyme method (d), (a) a method using α-amylase (JP-A-3-176000, JP-A-4-94698, etc.) and (b) sarcosine oxidase are used. (Japanese Patent Laid-Open No. 9-1872)
No. 96). Among them, the method (a) is a method in which inactivated α-amylase is reactivated by chloride ions, a sugar substrate is decomposed, and the decomposed products are measured to measure chloride ions in a body fluid. However, it is currently actually used in the field of clinical tests, coulometric titration,
Compared to the ion electrode method and the colorimetric method, they are superior in accuracy, simplicity, and analysis efficiency.

On the other hand, in recent years, liquefaction reagents have become mainstream in the clinical examination field due to demands for improving accuracy and stability, reducing costs, and saving labor. In the past, those that were unstable in the liquid state, such as enzymes, were circulated in the lyophilized state, and were dissolved in the attached dissolving solution at the time of use and used for measurement. By stabilizing the component enzymes by optimizing, etc., it is possible to distribute in the liquid state, so that the production side can save the time of lyophilization, and also the labor of dissolving at the clinical test site,
It has greatly contributed to labor savings related to clinical tests.

As described above, the electrolyte measurement method using α-amylase is superior in terms of simplicity, accuracy, and the like, among various conventional measurement methods. In this method, α-amylase is usually used in an inactivated form after removing calcium or chloride ions necessary for the expression of the activity to be measured. In addition, in this method, a chelating agent is prescribed in the measurement reagent for the purpose of suppressing a blank reaction, adjusting the quantification as a competitive inhibitor, or masking a similar contaminating ion outside the measurement object. . However, such an inactivated α-amylase is unstable in the presence of a chelating agent, and thus has a problem that it cannot be stored for a long time in a solution state.

On the other hand, as a method for stabilizing an inactivated α-amylase in the presence of a chelating agent, oligosaccharides such as maltose and α-cyclodextrin, or a mixture thereof are conventionally useful. This is known (JP-A-6-113894). However, this method can withstand short-term (1 to 2 months) low-temperature (2 to 8 ° C.) storage, but can also withstand long-term storage or actual operation at room temperature (18 to 37 ° C.). Stability is still not enough.

[0013] Also, as a problem derived from liquefaction,
There is an influence of contamination with α-amylase derived from a substance other than the measurement sample from the outside. This is because, for example, when human saliva or the like is accidentally mixed into the test solution, the substrate is decomposed by α-amylase contained in the saliva, and the reagent blank increases, the measurement sensitivity decreases, and the quantitativeness and precision are reduced. Is a problem. In particular, liquid reagents that are liable to be mixed during the production or use of a measurement test solution and that can be stored for a long period of time even in the presence of a slight amount of α-amylase derived from sweat or saliva affect the reagent performance. Given this, stabilization against α-amylase is required.

As to the problem of α-amylase contamination from substances other than the sample to be measured, as described in JP-A-6-277096, p-nitrophenyl-β- Galactosyl-α-
There is known a method of stabilizing maltopentaoside by converting it into an aqueous solution having a pH in the range of 2.0 to 5.5. However, although this method can stabilize the α-amylase substrate itself, it is not sufficient because the pH conditions of each test solution are restricted.

[0015]

As described above, regarding the method for measuring an electrolyte using α-amylase, a high solution stability that can withstand distribution as a liquid reagent which is a mainstream reagent for clinical testing today. Has not been achieved yet. The present invention, in view of the current situation as described above,
An object of the present invention is to provide a reagent composition for measuring an electrolyte used in an enzyme assay having excellent stability, accuracy and quantitativeness.

[0016]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, have determined that a measuring reagent is composed of two reagents and reversibly regenerated by a chelating agent and an electrolyte caused by poor stability. By separately formulating the inactivating α-amylase to be activated, long-term solution stability from room temperature to room temperature, which is a practical use level, can be achieved without using a stabilizing agent as in the past. And further, the configuration of the two reagents is changed at least in the first reagent to (a)
An α-amylase substrate, (b) a chelating agent, a second reagent, (c) an inactivated α-amylase which is reactivated reversibly by an electrolyte, and (d) a first reagent
The present inventors have found that inclusion of a substance having an inhibitory activity on α-amylase can avoid the influence of contaminating α-amylase, and completed the present invention.

That is, the present invention has the following configuration. (1) In a reagent composition for measuring an electrolyte comprising two reagents using α-amylase, an inactivated α-amylase which is reactivated reversibly by an electrolyte and a chelating agent are separately formulated. An electrolyte measurement reagent composition. (2) Inactivated α which contains (a) α-amylase substrate and (b) chelating agent in the first reagent and (c) reversibly reactivated by the electrolyte in the second reagent
-The reagent composition for measuring an electrolyte according to (1), which is formulated to contain amylase. (3) The reagent composition for measuring an electrolyte according to (1) or (2), wherein the first reagent comprises (d) a substance having an inhibitory activity on α-amylase. (4) The reagent for measuring an electrolyte according to any one of (1) to (3), wherein the α-amylase substrate is 2-chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside. Composition. (5) a substance having an inhibitory activity on α-amylase,
5-bromo-5-nitro-1,3-dioxane, 2-chloroacetamide, 2-hydroxypyridine-N-oxide, imidazolidinyl urea, N-methylisothiazolone, 5-chloro-2-methyl-4-isothiazoline- The reagent composition for measuring an electrolyte according to any one of (1) to (4), which is at least one selected from the group consisting of 3-one and N-ethylmaleimide. (6) The reagent composition for measuring an electrolyte according to any one of (1) to (5), wherein the chelating agent is 1,2-bis (o-aminophenoxy) ethanetetraacetic acid. (7) The first reagent has a pH of 7 or more and the second reagent has p
The reagent composition for measuring an electrolyte according to any one of (1) to (6), which is H6 to H7. (8) The electrolyte is a calcium ion (1) to (7)
5. The reagent composition for measuring an electrolyte according to any one of the above. (9) The reagent composition for measuring an electrolyte according to any one of (1) to (7), wherein the electrolyte is a chloride ion.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The reagent composition for measuring an electrolyte of the present invention is decomposed by utilizing the fact that α-amylase is activated by calcium or chloride ions which are activators of α-amylase. This is to measure the electrolyte in the sample by measuring the sugar substrate, and the following measurement principle can be considered. Here, the electrolyte refers to a substance that dissolves in water or another solvent to cause the solution to conduct ions, and specifically, calcium ions, chloride ions, magnesium ions, barium ions, manganese ions, Refers to zinc ions and the like.

(1) Method using maltooligosaccharide as a substrate Maltotetraose, maltopentaose,
Using maltohexaose or the like, activated α-amylase, and further, by the action of a follow-up enzyme such as α-glucosidase, glucose is released from these substrates, and the amount of glucose is measured. Ask for. As a method for measuring the produced glucose, there are a glucose oxidase-peroxidase method, a hexokinase-glucose-6-phosphate dehydrogenase method and the like.

In the glucose oxidase-peroxidase method, glucose oxidase is allowed to act on glucose, and the produced hydrogen peroxide is oxidized and condensed with a oxidative coloring substance such as phenol and a coupler in the presence of peroxidase to lead to a quinone dye, and the absorbance is measured. How to The hexokinase-glucose 6 phosphate dehydrogenase method converts glucose to glucose-6-phosphate by hexokinase, followed by glucose-6-phosphate.
Glucose in the presence of NAD ⁺ or NADP ⁺
NADH by reacting 6-phosphate dehydrogenase
Alternatively, it is a method of measuring the rate of increase reaction of NADPH.

(2) A method using a derivative in which a phenyl group, a naphthyl group, or a derivative thereof is bonded as an aglycone to the reducing end of maltooligosaccharide as a substrate: p-nitrophenylmaltopentaoside, p-nitrophenylmaltohexao Sid, p-nitrophenylmaltoheptaoside, 2,4-dichloronitrophenylmaltopentaoside, 2-chloro-4-nitrophenylmaltotrioside, 2-chloro-4-nitrophenylmaltopentaoside, etc. as substrates By using activated α-amylase and, if necessary, by following an enzyme such as α-glucosidase to release aglycone from these substrates and optically measuring the amount of released aglycone. Determine the mass of the electrolyte such as calcium.

(3) The hydroxyl groups at the 4- and 6-positions of glucose at the non-reducing end of a maltooligosaccharide derivative in which a phenyl group, a naphthyl group, or a derivative thereof are bonded to the reducing end of the maltooligosaccharide as an aglycone Method Using Modified Derivative as Substrate This is a method according to (2) except that a non-reducing terminal glucose is modified with a halogen, a glucopyranosyl group or the like (for example, JP-A-60-237998).
Or a substrate of the type in which the hydroxyl groups at the 4- and 6-positions are substituted with an alkyl group, an alcoyl group or a phenyl group (for example, JP-A-60-54395, JP-A-1-157996) or Substrates of the type in which the hydroxyl groups at the 6-position and 6-position are substituted with a β-galactopyranosyl group (see, for example, JP-A-3-26459)
No. 6, JP-A-6-315399).

Among the known measuring methods described above, the measuring method shown in the above (3) is excellent in principle. Among them, 2-chloro-4-nitrophenyl-4-o-β-D
In the method using -galactopyranosyl-α-maltoside, since the non-reducing end is modified, there is no increase in the reagent blank due to degradation by endogenous α-glucosidase or the like, and no follow-up enzyme is required. It is particularly preferable because it has the merit of low cost and high sensitivity due to high substrate affinity of α-amylase.

As an embodiment of the present invention, the measurement of calcium ion is described as follows. First, a substance having an inhibitory activity on α-amylase, a chelating agent, and 2-chloro-substrate as a substrate of α-amylase are used as a first reaction. -4
-Nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside is reacted with a reagent composition for measuring an electrolyte, followed by a second reaction in which the electrolyte is reversibly reactivated by the electrolyte. By reacting the reagent composition for measuring electrolyte containing type α-amylase, the amylase obtained and reactivated by obtaining calcium in the sample decomposes the substrate and measures 2-chloro-4-nitrophenol released. Then, the amount of calcium in the sample is determined.

In the present invention, the substance having an inhibitory activity on α-amylase is a substance that binds to a specific site of α-amylase and reduces the reaction rate. It refers to a substance having an inhibitory action, for example, a substance that acts or binds to a thiol group, amino group, or the like constituting a protein. Specifically, 5-bromo-5-nitro-1,3
-Dioxane, 2-chloroacetamide, 2-hydroxypyridine-N-oxide, imidazolidinyl urea, N-methylisothiazolone, 5-chloro-2-methyl-4-isothiazolin-3-one, N-ethylmaleimide, p- Examples include, but are not limited to, chloromercury benzoic acid and β-bromoethylamine, and a plurality of these may be used in combination. The concentration of the substance having an inhibitory activity on the α-amylase in the reagent composition is not particularly limited, but may have an effect on an inactivated α-amylase which is reactivated reversibly by an electrolyte contained in the reagent composition. Considering, 0.
It is preferably used in the range of 01 to 100 mM.

In the present invention, the α-amylase can be of any origin of microorganisms, plants or animals, but is preferably of animal origin, such as porcine pancreas, human pancreas, Α-amylase derived from human saliva is exemplified. The α-amylase used in the present invention needs to be inactivated by desalting. Then, the inactivated α-amylase actually involved in the measurement is activated α-amylase by obtaining an electrolyte such as calcium or chloride ions in the sample as described above, and becomes α-amylase.
It must react with the amylase substrate and be reactivated reversibly by the electrolyte. The desalting treatment is preferably performed by a method such as dialysis, ultrafiltration, ion exchange, column removal, and electrodialysis. The concentration of the inactivated α-amylase which is reactivated reversibly by the electrolyte in the reagent composition is preferably 0.1 to 1 in the reaction solution.
It is used in the range of 000 IU / ml.

With respect to the specificity for selecting a specific ion from a plurality of ions, each concentration range of interfering ions such as ions other than the object to be measured is estimated, and the performance of the sample to be measured is also taken into consideration. Then, by coexisting a chelating agent specific to the interfering ion, the activation of α-amylase by the interfering ion is suppressed, or
This can be achieved by a method in which a sufficient amount of ions which can be interfering ions are previously present in the composition so that the interfering ions are not disturbed at the concentration level of the interfering ions in the sample. To illustrate a specific embodiment, when measuring calcium ions, chlorine ions and the like become interfering ions,
For example, a compound containing chlorine, such as sodium chloride, is allowed to coexist in the reagent composition at a level not affected by the measurement. When measuring chloride ions, calcium ions and the like can be interfering ions, and thus a method of adding a chelating agent that traps calcium ions in the sample can be used.

In the present invention, examples of the chelating agent include ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, 1,2-bis (o-aminophenoxy) ethanetetraacetic acid, trans-1,2-diaminocyclohexanetetraacetic acid and these. And the like. Among them, 1,2-bis (o-aminophenoxy) is preferred from the viewpoint of metal selectivity in the vicinity of neutral pH which is the optimum pH in the present invention.
Ethanetetraacetic acid is particularly preferred. The role of the chelating agent in the electrolyte measurement reagent composition includes suppressing a blank reaction as described above, adjusting quantification as a competitive inhibitor, or masking a similar contaminating ion outside the measurement target. No. The chelating agent is preferably contained in the reaction solution at 0.01 to 10 mM, more preferably 0.1 to 1 m.
Used in the range of M. Further, a plurality of the chelating agents shown above may be used in combination.

The substrate of the α-amylase according to the present invention is not limited at all as described above. However, 2-chloro-4-nitrophenyl is advantageous because it does not require a follow-up enzyme and is low in cost. Maltotrioside, 2-chloro-4-nitrophenyl 4-o-β-D-
Galactopyranosyl maltoside is preferred. further,
Since the non-reducing end is modified, there is an advantage that there is no increase in the reagent blank due to degradation by endogenous α-glucosidase and the like, and that α-amylase has a higher substrate affinity and thus has higher sensitivity. Chloro-
4-Nitrophenyl 4-o-β-D-galactopyranosyl maltoside is more preferably used. The concentration of the substrate in the reagent composition is preferably 0.05 to
It is used in the range of 50 mM, more preferably in the range of 0.1 to 2 mM.

Further, the reagent composition for measuring an electrolyte of the present invention comprises:
Since it is necessary to perform measurement in two steps of the first reaction and the second reaction, it is necessary to use a so-called two-reagent system constituted by two reagents. Then, an inactivated α-amylase which is reactivated reversibly by a chelating agent and an electrolyte is separately formulated. By separately formulating the chelating agent and the inactivated α-amylase, long-term solution stability of the inactivated α-amylase can be obtained. Further, by setting the final pH of the electrolyte measuring reagent composition in the range of 5.0 to 8.0,
It is possible to control the rate of the sugar decomposition reaction of α-amylase itself and to extend the measurement range.

After the addition of the second reagent, a zero-order reaction capable of measuring the rate can be obtained more quickly, or (a) α
An embodiment comprising an amylase substrate and (b) a chelating agent, and wherein the second reagent is formulated to contain (c) an inactivated α-amylase which is reversibly reactivated by an electrolyte. . Further, from the viewpoint of avoiding the influence of the contamination of α-amylase derived from a substance other than the sample to be measured from the outside, the above-mentioned first reagent contains (d) a substance having an inhibitory activity on α-amylase. Is more preferable.

On the other hand, the optimum pH of the stability of α-amylase is around 6 to 8 neutrality, and the optimum pH of the chelating agent is on the alkaline side. It is considered to be preferable to prepare in the range of ~ 8, but in order to obtain the performance such as quantitativeness at the same time, it is also possible to formulate the first reagent and the second reagent so that the pH becomes the optimal reaction when the second reagent is mixed. it can. Preferably, the reagent containing the chelating agent is p
H7 or more, more preferably pH 7 to 9, the pH of the reagent containing an inactivated α-amylase which is reactivated reversibly by the electrolyte is adjusted to pH 6 to 7, and the buffer concentration or the mixing ratio of these two reagents is adjusted. It is formulated to have an appropriate pH. The method for maintaining the reagent pH is not particularly limited as long as it is a known method, but a buffer is generally used. Examples of the buffer include a good buffer, a Tris buffer, a phosphate buffer and the like. First reagent,
The buffer in each of the second reagents is preferably 10-50
It is used at a concentration of 0 mM, more preferably 50-300 mM.

In addition, the reagent composition for measuring an electrolyte of the present invention may be used as a regulator substrate separately from the α-amylase substrate (so-called mediator substrate) for the purpose of suppressing a reagent blank, improving quantitative performance, and the like. If necessary, a combination of two substrates or substrates and products thereof having different final optical measurement methods can be used to compete with each other for the glycolysis reaction and reduce the apparent substrate affinity. For example, maltooligosaccharides, or maltooligosaccharides in which the non-reducing terminal glucose is modified, or a substrate selected from maltooligosaccharides in which a non-chromogenic source group is bonded to the reducing terminal glucose, and the main terminal reaction, the reducing terminal glucose, is a chromogenic source. Attach a group, or
Further, α for a substrate having a substituent bonded to the non-reducing end
-Adjust the reaction rate of amylase. Among them, maltooligosaccharides with non-reducing terminal glucose modified endogenous α-
It is preferably used because it is not affected by glucosidase degradation and does not affect the measured value.

Examples of the maltooligosaccharides include maltooligosaccharides having a glucose number of 2 to 7, such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Examples of maltooligosaccharides having a non-reducing terminal glucose modified include galactosyl maltose, galactosyl triose, galactosyl tetraose, and galactosyl pentaose.
4-dichlorophenyl-α-D-maltotrioside,
2,4-dichlorophenyl- (α or β) -D-maltopentaoside, 2,4-dichlorophenyl- (α or β) -D-maltotrioside and the like can be mentioned. The concentration of the maltooligosaccharide used is preferably 0.01 to 250 mM in the reaction solution, more preferably 0.1 to 2 mM.
00 mM, and can be used in both the first and second reagents.

The reagent composition for measuring an electrolyte of the present invention may further comprise, if necessary, a selective binder for the interference electrolyte or the electrolyte to be measured in order to avoid the influence of an interference electrolyte such as ionophore and crown ether, or to adjust the sensitivity. Can be used. Examples of the selective binder include 18-crown-6 (manufactured by Merck) and Kryptofix 221 (manufactured by Merck) in addition to the above-described chelating agents. If necessary, preservatives, surfactants, antioxidants, protease inhibitors, and the like can be added in a range that does not affect the quantitativeness of the electrolyte to be measured.

The preservative is not particularly limited, and sodium azide having little effect on the stability of α-amylase,
Alternatively, cephem-based, penicillin-based, aminoglycoside-based, quinolone-based antibiotics and the like are preferably used, and these can be used alone or in combination.
As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or the like can be used alone or in combination.

When the electrolyte to be measured is calcium ion, it is preferable to add a halide of an alkali metal, for example, sodium chloride or potassium chloride at a concentration of 3 to 300 mM. When the electrolyte to be measured is chloride ion, a divalent cation, for example, calcium, magnesium, barium, zinc or the like can be added at a concentration of 0.01 to 200 mM.

Examples of the antioxidant include ascorbic acid and salts thereof, saccharides such as sorbose, and catalase. Protease inhibitors include phenylmethylsulfonyl fluoride and the like.

In the present invention, the inactivated α-amylase which is reactivated reversibly by the electrolyte is converted into an activated α-amylase by obtaining an electrolyte such as calcium or chloride ions in the sample as described above. The reaction can be measured based on the above-described measurement method by utilizing the reaction with the α-amylase substrate. For example, in the case of measuring calcium, as a first reaction, a substance having an inhibitory activity on α-amylase, a chelating agent, and 2-chloro-4-nitrophenyl-4 as a substrate of α-amylase are added to the sample.
-O-β-D-galactopyranosyl-α-maltoside is reacted with a first reagent, followed by a second reaction containing an inactivated α-amylase which is reversibly reactivated by an electrolyte. By reacting the two reagents, 2-chloro-4-nitrophenol is produced by the reaction of the amylase reactivated depending on calcium in the sample.

Since 2-chloro-4-nitrophenol itself absorbs at around 400 nm, it is 40
The change in absorbance around 0 nm is measured, and the concentration of calcium in the sample is determined using the absorbance of a sample of known concentration as a control.
As the method for measuring 2-chloro-4-nitrophenol, any of a rate method for continuously following the amylase reaction and an end point method for measuring after stopping the reaction after reacting for a certain period of time can be applied.

As an embodiment of the present invention, the measurement of chloride ions is described as follows. As a first reaction, a substance having an inhibitory activity on α-amylase, a chelating agent and α
Reacting a first reagent containing 2-chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside as an amylase substrate, followed by a reversible reaction with an electrolyte as a second reaction By reacting a second reagent containing an inactivated α-amylase to reactivate, 2-chloro-4-nitro is formed by the reaction of the amylase reactivated depending on chloride ions in the sample. Phenol is measured to determine the amount of chloride ions in the sample.

[0042]

The present invention will be described in more detail with reference to the following examples. Note that the present invention is not limited to these examples.

Example 1 A calcium measuring reagent having the following composition was prepared, and a second reagent containing α-amylase was stored at 35 ° C. for 14 days.
The α-amylase activity in the test solution on the 5th, 10th, and 14th days immediately after preparation was measured using a commercially available α-amylase activity measuring reagent (Diacolor AMY Neolate: manufactured by Toyobo Co., Ltd.), and the residual activity was measured. (%) Was determined. Table 1 shows the results.

(1) Composition of First Reagent Tris-HCl buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0 0.05% 2-chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 0.9 mM

(2) Composition of the second reagent Good buffer (pH 6.0) 300 mM NaCl 200 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0.05% inactivated α-amylase (derived from pig pancreas) 2 IU / ml

Comparative Example 1 A calcium measuring reagent having the following composition was prepared, and the first reagent containing α-amylase was stored at 35 ° C. for 14 days. Residual activity (%) after storage at 35 ° C. as in Example 1. I asked. Table 1 shows the results.

(1) Composition of First Reagent Tris-HCl buffer (pH 7.1) 50 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0.05 % NaCl 200 mM Inactivated α-amylase (porcine pancreas) 2.1 IU / ml

(2) Composition of second reagent Good buffer (pH 6.0) 300 mM NaCl 200 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0.05% 2-chloro-4-nitrophenyl-4-o-β -D-galactopyranosyl-α-maltoside 1.8 mM

[0049]

[Table 1]

As shown in Table 1, the residual activity of α-amylase of Comparative Example 1 was 39.4%, whereas that of Example 1 was 97.2%.
%, Indicating little deterioration and good stability.

Example 2 The same calcium measurement reagent as in Example 1 was stored at 35 ° C. for 14 days, and together with immediately after the preparation, purified water, 10 m
The g / dl standard solution and the calcium acetate aqueous solution (50 mg / dl in terms of Ca ion) were measured at 10 levels of dilution series, and the high value linearity was examined.

Measurement method of calcium amount 180 μl of the first reagent was added to 3.5 μl of the sample, and after preliminarily heating for 5 minutes, 90 μl of the second reagent was further added to start the reaction, and 2 minutes after the addition of the substrate reagent solution, The change in absorbance per minute for 3 minutes was determined, and the amount of calcium in the sample was determined based on two calibration curves with purified water and a 10 mg / dl calcium standard solution. The measuring device was Hitachi 717
Using a type 0 automatic analyzer, the measurement wavelength is 405
nm, the auxiliary wavelength was 546 nm, and the measurement temperature was 37 ° C.

Comparative Example 2 The same calcium measuring reagent as in Comparative Example 1 was stored at 35 ° C. for 14 days, and the high value linearity was examined in the same manner as in Example 2 together with immediately after the preparation.

From FIGS. 1 and 2, the linearity of Comparative Example 2 is significantly increased at a high value, whereas the linearity of Example 2 hardly rises, good linearity is obtained, and stability is improved. It was confirmed that there was.

Example 3 As substances having the following composition having inhibitory activity against α-amylase, (A) 5-bromo-5-nitro-1,3 dioxane, (A) 2-chloroacetamide, (C) 2- Hydroxypyridine-N-oxide, (d) imidazolidinyl urea, (e) N-methylisothiazolone, (f)
Contains 5-chloro-2-methyl-4-isothiazolin-3-one, (g) 7 reagents each containing 0.05% of N-ethylmaleimide, and (h) a substance having an inhibitory activity against α-amylase. A total of eight calcium measurement reagents were prepared, and the reagents were mixed with human saliva-derived α-amylase in the reagents at 3 IU / L and stored at 35 ° C. for 14 days. In addition, each reagent was similarly stored with respect to a reagent not containing α-amylase. The measurement was performed in accordance with the method for measuring the amount of calcium in Example 2, and purified water was measured as a sample, and the initial absorbance of the reagent at 35 ° C. immediately after preparation for 5, 10, and 14 days (the starting point of the photometry starting point when measuring the purified water) Absorbance). Table 2 shows the results.

(1) Composition of the first reagent Tris-HCl buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0 0.05% 2-chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 0.9 mM Substance having inhibitory activity against α-amylase 0.05%

(2) Composition of second reagent Good buffer (pH 6.0) 300 mM NaCl 200 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 0.05% inactivated α-amylase (derived from porcine pancreas) 2 IU / ml

Comparative Example 3 A calcium measuring reagent having the composition shown in Comparative Example 1 was prepared, and a similar sample was measured in the same manner as in Example 3, and immediately after preparation, at 35 ° C. for 5, 10, and 14 days. The initial absorbance of each reagent was determined. Table 2 shows the results.

[0059]

[Table 2]

From Table 2 above, the initial absorbance of the α-amylase-contaminated reagent of Comparative Example 3 was clearly higher than that of the uncontaminated reagent, whereas in Example 3, the initial absorbance was increased for any of the reagents. Is suppressed. Above all, it was confirmed that the increase in the initial absorbance was remarkably improved in a reagent containing a substance having an inhibitory activity on α-amylase in the first reagent.

Example 4 A calcium measuring reagent having the following composition was stored at 40 ° C., and the reagent was used 1, 4 and 7 days after immediately after preparation.
0mg / dl standard solution and commercially available serum monitrol I
X and monitrol IIX were measured, and the sensitivity of 10 mg / dl standard solution and the daily change in serum value were determined as relative values (%) with the control immediately after preparation as a control. The results are shown in Tables 3 and 4.

(1) First Reagent Composition Tris-HCl buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octylphenyl Ether 0.05% 2-Hydroxypyridine-N-oxide 0.05% 2-Chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 0.9 mM (2) Second Reagent composition Good buffer (pH 6.5) 300 mM NaCl 200 mM polyoxyethylene octyl phenyl ether 0.05% inactivated α-amylase (derived from pig pancreas) 4.2 IU / ml

Comparative Example 4 A calcium measuring reagent having the following composition was used in Example 4
The same study was conducted. The results are shown in Tables 3 and 4. (1) Composition of first reagent Tris-HCl buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octyl phenyl ether 05% 2-hydroxypyridine-N-oxide 0.05% Inactivated α-amylase (derived from porcine pancreas) 2.1 IU / ml (2) Second reagent composition Good buffer (pH 6.5) 300 mM NaCl 200 mM poly Oxyethylene octyl phenyl ether 0.05% 2-chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 1.8 mM

Example 5 Example 4 for a calcium measuring reagent having the following composition:
The same study was conducted. The results are shown in Tables 3 and 4.

(1) Composition of First Reagent Tris-HCl Buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-Bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM Galactosyl maltose 2.3 mM Polyoxyethylene octylphenyl Ether 0.05% 2-Hydroxypyridine-N-oxide 0.05% 2-Chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 0.9 mM

(2) Composition of Second Reagent Good Buffer (pH 6.5) 300 mM NaCl 200 mM Polyoxyethylene octylphenyl ether 0.05% Inactivated α-amylase (derived from human saliva) 12 IU / ml

Comparative Example 5 Example 4 for a calcium measuring reagent having the following composition:
The same study was conducted. The results are shown in Tables 3 and 4.

(1) Composition of First Reagent Tris-HCl buffer (pH 7.1) 50 mM NaCl 200 mM 1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM galactosyl maltose 2.3 mM polyoxyethylene octylphenyl Ether 0.05% 2-hydroxypyridine-N-oxide 0.05% Inactivated α-amylase (derived from human saliva) 6 IU / ml

(2) Composition of Second Reagent Good Buffer (pH 6.5) 300 mM NaCl 200 mM Polyoxyethylene octyl phenyl ether 0.05% 2-chloro-4-nitrophenyl-4-o-β-D-galactopyra Nosyl-α-maltoside 1.8 mM

[0070]

[Table 3]

[0071]

[Table 4]

Table 3 shows that the sensitivity of the 10 mg / dl standard solution changes with time in Comparative Examples 4 and 4 and in Comparative Examples 5 and 5 that the sensitivity of Examples 4 and 5 after storage at 40.degree. Turned out to be small. In addition, a comparison between Examples 4 and 5 showed that the sensitivity of the reagent using human saliva was smaller than that of α-amylase derived from porcine pancreas, and the storage stability was superior. From Table 4, it can be seen that the relative values (%) of monitrol IX in Comparative Examples 4 and 5 in the daily changes in serum values
, A decrease was observed, and monitrol IIX showed an increase, whereas in Examples 4 and 5, almost no change was observed.

[0073]

As described above, the reagent composition for measuring an electrolyte according to the present invention comprises two measuring reagents, and has an inertness which is reactivated reversibly by a chelating agent and an electrolyte caused by poor stability. By separately formulating the modified α-amylase, long-term solution stability from a low temperature to a practical use level at room temperature can be obtained without using a conventional stabilizer unnecessarily. In addition, the inactive α-amylase which is reactivated reversibly by (a) α-amylase substrate and (b) chelating agent in the first reagent and (c) electrolyte in the second reagent at least in the composition of the two reagents. And (d) α in the first reagent.
-By containing a substance having an inhibitory activity on amylase, the influence of contaminating α-amylase can be avoided, so that it is possible to provide a reagent composition for measuring an electrolyte which is excellent in stability, accuracy and quantitativeness.

[Brief description of the drawings]

FIG. 1. In Example 2, immediately after preparation and at 35 ° C., 14 ° C.
The results of measuring 10 levels of an aqueous solution of ５０50 mg / dl using the reagents stored for days, the dilution level on the X-axis,
It is the figure which plotted with calcium concentration (mg / dl) on the Y-axis.

FIG. 2 shows the results obtained in Comparative Example 2 immediately after preparation and at 35 ° C. and 14 ° C.
The results of measuring 10 levels of an aqueous solution of ５０50 mg / dl using the reagents stored for days, the dilution level on the X-axis,
It is the figure which plotted with calcium concentration (mg / dl) on the Y-axis.

Claims (9)

[Claims] 1. An electrolyte measuring reagent composition comprising two reagents using α-amylase, wherein an inactivated α-amylase reversibly reactivated by an electrolyte and a chelating agent are separately formulated. And a reagent composition for measuring an electrolyte. 2. An inactivated α-amylase containing (a) an α-amylase substrate and (b) a chelating agent in a first reagent, and (c) reversibly reactivated by an electrolyte in a second reagent. The reagent composition for measuring an electrolyte according to claim 1, wherein the reagent composition is formulated to contain -ze. 3. The reagent composition for measuring an electrolyte according to claim 1, wherein the first reagent comprises (d) a substance having an inhibitory activity on α-amylase. 4. The method according to claim 1, wherein the α-amylase substrate is 2-chloro-4-.
The reagent composition for measuring an electrolyte according to any one of claims 1 to 3, which is nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside. 5. The substance having an inhibitory activity on α-amylase is 5-bromo-5-nitro-1,3-dioxane, 2-chloroacetamide, 2-hydroxypyridine-N-oxide, imidazolidinyl urea, The at least one member selected from the group consisting of N-methylisothiazolone, 5-chloro-2-methyl-4-isothiazolin-3-one and N-ethylmaleimide.
5. The reagent composition for measuring an electrolyte according to any one of 4. 6. The reagent composition for measuring an electrolyte according to claim 1, wherein the chelating agent is 1,2-bis (o-aminophenoxy) ethanetetraacetic acid. 7. The reagent composition for measuring an electrolyte according to claim 1, wherein the first reagent has a pH of 7 or more, and the second reagent has a pH of 6 to 7. 8. The reagent composition for measuring an electrolyte according to claim 1, wherein the electrolyte is a calcium ion. 9. The method according to claim 1, wherein the electrolyte is chloride ion.
The reagent composition for measuring an electrolyte according to any one of the above.