JPH08160049A - Potassium ion measuring reagent composition and test piece - Google Patents

Abstract

PURPOSE: To quantitatively determine potassium ion at least in the range of 0-50mmol/l, with no dilution operation and binder agent, by using nicotinamide adenine dinucleotide synthesized enzyme. CONSTITUTION: This reagent composition is a composition for measuring potassium ions in a sample liquid, oxidation nicotinamide adenine dinucleotide synthesized enzyme[EC6. 3. 5. 1.], adenosine triphosphate, bivalent metal ion, deamide nicotinamide adenine dinucleotide, amino group donator, dehydrogenase and matrix corresponding to it, tetrazolium salt and diaphorase are contained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for measuring potassium ion and a test piece for measuring potassium ion containing the composition, more specifically, for example, in a clinical test, in a body fluid, particularly serum, plasma or urine. The present invention relates to a reagent composition for measuring potassium ion contained in and a dry test piece containing the same.

[0002]

2. Description of the Related Art Measuring ions contained in electrolytes such as potassium ions is important for judging the homeostasis of a living body. Especially, potassium ions in body fluid measured in clinical tests are It gives very useful clues to know the function of organs, such as renal function.

Known ion measuring methods include a flame photometric method, an atomic absorption photometric method, and an ion selective electrode method. Further, in recent years, a so-called “enzyme method”, which utilizes an enzymatic reaction of an enzyme that is activated in an ion concentration-dependent manner in the presence of an ion to be measured, has been attracting attention. As an example of the enzymatic method, when the target ion for measurement is potassium, clinical chemistry (Clin. Chem.), 35/5, 8
17-820 (1989), a method utilizing pyruvate kinase is also described in European Patent EP-B-0 286 03.
9 describes a method utilizing pyruvate kinase, glycerol dehydrogenase or acetaldehyde dehydrogenase.

[0004]

Although the flame photometric method and the atomic absorption photometric method are excellent in terms of accuracy, they require a large-scale dedicated measuring instrument and are very laborious for pretreatment of reagents and specimen samples. It is not suitable for processing a large number of specimens because it takes much time and the analysis takes time. Further, although the ion-selective electrode method is easy to handle the electrode itself, an electrode measuring device dedicated to electrolyte measurement is required, and only the electrolyte can be measured by the measuring device. For example, the ion-selective electrode method cannot be combined with a test piece for multi-item inspection for checking the degree of color development such as glucose or protein.

Known methods for measuring potassium ions by an enzyme method in a liquid system include a pyruvate kinase method, a glycerol dehydrogenase method, an acetaldehyde dehydrogenase method and the like, all of which are affected by coexisting ions such as sodium ions. That is, it is interfered. Therefore, various binders such as cryptant, coroland, podanto, crown ether, spherand, hemispherand, calixarene, and combinations thereof, naturally occurring ionophores, cyclic peptides, complexons, chelating agents and their derivatives are added, The effect of coexisting co-existing ions has to be avoided and the method is very cumbersome.

[0006] In the measuring method using the enzyme method, the object to be measured is a liquid, and in most cases is undiluted body fluid such as urine or blood. For example, about 4 mmol of potassium ion is contained in the serum of a healthy person. / L included. However, the above-mentioned enzyme is too concentrated at such a potassium ion concentration, so that the activity of the enzyme is saturated and the quantitative property is lost. In order to avoid this and to widen the target measurement range of potassium ion, a binder is added in the reaction of the liquid system.

When various components including an enzyme are to be prepared as a dry composition, the stability of the enzyme is generally remarkably lowered when the activating ion is removed by making the enzyme coexist with a binder, so that various drying methods are required. The preparation of a dry composition requiring steps is very difficult. Furthermore, it is difficult to keep the detection sensitivity stable because of changes in the enzyme concentration and the binder concentration. Thus, it can be said that it is almost impossible to apply the above enzyme together with the binder to the dry composition while maintaining the stability.

Interfering ions when measuring potassium ions are mainly sodium ions, but pyruvate kinase is also activated by ammonium ions, so it is necessary to eliminate the influence of ammonium ions. Clinical Chemistry, 35/5, 817-820 (198)
In 9), glutamate dehydrogenase [EC1.
4.1.4] is used to eliminate the endogenous ammonium ion.

When the method for determining potassium ion by the enzyme method is applied to a dry composition, since the natural polymer commonly used in the dry composition contains a large amount of potassium, the enzyme is affected by the potassium and the measurement results make worse.

[0010]

In view of the above-mentioned drawbacks of the prior art, as a result of earnest research to develop a simpler method, nicotinamide adenine dinucleotide (NA
D) The synthase is far more suitable than pyruvate kinase, glycerol dehydrogenase or acetaldehyde dehydrogenase disclosed in European Patent EP-B-0 286 039 when applied to a dry composition for determination of potassium ion. The inventors have found out and completed the present invention.

According to the first aspect of the present invention, there is provided a composition for measuring potassium ion in a sample solution, which comprises an oxidized NAD synthase [EC6.3.5.1. ], Adenosine triphosphate (ATP), divalent metal ion, deamide N
Provided is a reagent composition for measuring potassium ion, which comprises AD, an amino group donor, a dehydrogenase and a substrate corresponding thereto, a tetrazolium salt and diaphorase.
According to a second aspect, it comprises a support, a reagent layer formed thereon, and a spreading layer formed on the reagent layer,
The oxidized NAD synthase [EC 6.3.5.1. ], ATP, divalent metal ion, deamidated NAD, amino group donor, dehydrogenase and its corresponding substrate, tetrazolium salt, diaphorase, high molecular polymer and buffer for measuring potassium ion.

The determination of potassium ion in the present invention is as follows.
It is carried out by the following series of reaction systems. As can be understood from the above reaction system, NAD synthase
It catalyzes the reaction of (1).

As the NAD synthase [EC6.3.5.1], those derived from bacteria or yeast, and those derived from higher animals are known. The enzyme of any origin can be used in the present invention, but Bacillus stearothermophilu is excellent in heat resistance.
NAD synthase derived from s) is preferred.

The amino group donor may be any amino acid or various ammonium salts as long as NAD synthase can be used as a substrate. Preferred are glycine, ammonium chloride or ammonium sulfate.
The optimum environmental pH value for the enzymatic reaction of the reaction system is 8.0 to 1
Although it is around 0.0, preferably around 9.0, in the case of a dry test piece, since the ammonium salt evaporates under alkaline conditions, it is necessary to keep the pH near neutral in the layer containing the ammonium salt. The amount of amino group donor is sufficient, for example, K relative to the amino group donor of NAD synthase.
The density may be 20 times or more the m value. As described above, ammonium ion is contained in the reaction system from the beginning because it is an essential component, and a sufficient amount of ammonium ion has already been added. Ion detection is not affected at all.

The divalent metal ion acts as an activating component of NAD synthase. NAD synthase does not work without divalent metal ions. Examples of the divalent metal ion include magnesium ion and calcium ion.

ATP, a divalent metal ion, an amino group donor and a deamide NAD are components necessary for the enzyme reaction (1). NAD synthase is inactivated in the absence of these, but when NAD synthase and all of them are present in the same layer of the test piece, a trace amount of potassium ion contaminating causes the reaction during reagent preparation and storage. Happened,
It adversely affects the measured value. The problem is that NAD synthase and at least one of ATP, a divalent metal ion, an amino group donor and a deamidated NAD are separated between the layers of the test piece, and thus become substantially inactive during storage. It can be solved by the presence of the enzyme. Specifically, ATP, 2
The presence of at least one of a valent metal ion, an amino group donor, and a deamide NAD in the spreading layer
The NAD synthase can be present in the reagent layer in a substantially inactive state during storage. As a result, it is possible to produce a test piece which is not colored at all during storage and has excellent stability.

The reaction (2) is the NAD produced in the reaction (1).
(Oxidized nicotinamide adenine dinucleotide)
It is a reaction for converting to ADH (reduced nicotinamide adenine dinucleotide). Various dehydrogenases are known as enzymes that catalyze this reaction, and any of them may be used. A sufficient amount of the substrate together with the enzyme, for example, K for the amino group donor of NAD synthase is used.
The concentration is more than 20 times the m value. Just add it. A preferred combination of enzyme and substrate is a combination of 3α-hydroxysteroid dehydrogenase and its substrates sodium cholate, and isocitrate dehydrogenase and isocitrate.

The reaction (3) is a reaction for forming a formazan dye from the NADH produced in the reaction (2) and a tetrazolium salt. A wide variety of tetrazolium salts are commercially available, such as nitrotetrazolium blue, tetrazolium blue, and tetrazolium violet, and any of them may be used. It is preferable to use tetrazolium violet, which has excellent solubility and is highly stable against a reducing agent contained in a sample and light from the outside.

Diaphorase acts as a hydrogen messenger. Many kinds of diaphorase are also commercially available, but any one that acts on NADH may be used. Diaphorase is activated by various nonionic surfactants such as Triton X-100, Tween 20, Brij-58, etc. It is preferable to add about 5%, but it may not be added.

A brief summary of these reaction systems is as follows. (A) Deamide N in the presence of ATP and a divalent metal ion
NA from AD and amino group donor by NAD synthase
A reaction system in which an enzymatic reaction is specifically and concentration-dependently activated by coexisting potassium ions when D is produced. (B) An enzyme cycling reaction system containing various dehydrogenases for converting the NAD produced in (A) into NADH and a diaphorase for producing a formazan dye from the produced NADH and a tetrazolium salt.

The dry test strip containing the composition of the present invention has a reagent layer containing NAD synthase and optionally a part of other components on a support, and further has a reagent layer on the reagent layer. The developing layer containing the rest of the above.

This reagent layer is prepared by applying a liquid, in which an enzyme and reagents are substantially uniformly dissolved or dispersed in a hydrophilic polymer as a polymer binder, on the surface of a support to give a constant thickness. It is formed by drying. The thickness of the reagent layer is an appropriate thickness that does not interfere with the measurement, for example, 50 to 30.
It may be set to 0 mm. The coating and drying methods are performed by known methods.

The polymer used in the reagent layer must have a low potassium content, and specifically, synthetic polymers such as polyvinylpyrrolidone and polyvinyl alcohol satisfy this condition. These are used alone or in combination of two or more.

The optimum environmental pH value for the enzymatic reaction is 8.0 to
Since it is around 10.0, preferably around 9.0, this pH
The buffering agent that retains is appropriately selected from known buffering agents,
It may be contained in the layer.

As the spreading layer, a textile material is used after being washed with water. The fibrous material is impregnated with an aqueous solution containing a part of the reagent, dried, and then pressed or laminated on the reagent layer in a dry state or in a wet state again.

As the support, a resin which is usually used when producing a dry test piece of this type, for example, polyethylene terephthalate or the like processed into a strip shape may be used.

Quantitative analysis of potassium ions after applying the sample solution to the spreading layer of the test piece was carried out by measuring the rate of change of reflectance at the maximum absorption wavelength of formazan, and calibrating from the measurement result of potassium solution of known concentration. Calculate using the line.

[0028]

EFFECT OF THE INVENTION It is already known in the literature that NAD synthase is activated by potassium ions [Journal of Biological Chemistry (J. B.
iol. Chem. ) 247, 4794-4802 (197)
2) etc.). The inventors of the present invention have studied and examined it in more detail. As a result, NAD synthase has not only the property of being quantitatively activated in proportion to the concentration of potassium ion, but also surprisingly "(1) Potassium ion Has very high specificity for (2) the property of being proportionally activated even if the potassium ion concentration is very high, (3) the ammonium ion which is an interference ion in PK is essential for NAD synthase It is a component and has the property of not being affected even if it is present in a large amount. "

Because of these properties, the use of NAD synthase eliminates the need for binders such as cryptants and chelating agents as used in the invention of European Patent EP-B-0 286 039. Due to its high specificity for potassium, the effects of other coexisting ions (mainly sodium ions) can be easily avoided. Further, since ammonium ion is an essential component in the reaction system of the present invention, it is not necessary to consider the presence of ammonium ion in addition to sodium ion.

Further, since a very high concentration of potassium ion in the range of at least 0 to 50 mmol / l can be quantified without adding a binder, serum, plasma, urine containing high concentration of potassium ion, let alone With other liquids, it is possible to measure the potassium ion concentration without diluting or adding a binder.
The application to dry compositions is also very simple, as there is no need to dilute the sample. In addition, ATP, divalent metal ion,
N-containing at least one of the amino group donor and the deamidated NAD
By separating from the AD synthase, it is possible to provide a dry test strip having excellent storage stability.

Various polymers are used as binders in the production of dry test pieces, but a large amount of potassium ions are mixed in the natural polymer, which adversely affects the measurement results. This problem is solved by using a synthetic polymer containing no potassium ion, and it is possible to prepare a test piece having no background coloration. As a result, it can be incorporated in the same test piece as the test piece of the multi-item test for checking the degree of color development such as glucose and protein.

[0032]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Example 1 The following components were used in this example.

Potassium test specimen formulation : Reagent layer coating liquid component Tris-HCl buffer (pH 9.0) 50 mmol / l Isocitrate trisodium salt (Nacalai Tesque) 30 mmol / l ATP 5 mmol / l MgCl ₂ 1 mmol / l Triton X -100 0.2% (W / V) NAD synthase (Asahi Kasei) 15 KU / l Isocitrate dehydrogenase (Oriental yeast) 100 KU / l Diaphorase (Asahi Kasei) 20 KU / l Kollidone 90F (BASF) 8% ( W / V) (adjusted to pH 9.0 with sodium hydroxide solution)

Development layer impregnating solution component Tris-hydrochloric acid buffer solution (pH 7.5) 10 mmol / l tetrazolium violet (Sigma) 60 mmol / l Deamid NAD (Asahi Kasei) 30 mmol / l (NH ₄ ) ₂ SO ₄ 60 mmol / l Triton X-100 0.2% (W / V) Kollidon 90F 8% (W / V) (adjusted to pH 7.5 with sodium hydroxide solution)

After mixing the above-mentioned reagent layer coating liquid components, a support (polyethylene terephthalate white film. Thickness 1
25 μm. ) Was coated on the above to a thickness of 150 μm (wet thickness) and dried at 50 ° C. for 15 minutes. The mixed solution of the components of the spreading layer is coated on another support to a thickness of 50 μm, and the fiber material is placed on the coating film while the coating film is wet, and the components are absorbed by the fiber material. And dried at 50 ° C. for 15 minutes. Peel off the dried sample holding layer from the support and remove purified water to 1 m ²
Impregnate 20 ml per unit and press-bond on the reagent layer, then 50 ℃
And dried for 10 minutes.

The support laminated with the reagent layer and the spreading layer was cut with a cutting machine so that the test piece portion became 5 × 7 mm, and stuck to a stick-shaped white polyethylene terephthalate plate having a size of 5 × 70 mm and a thickness of 0.25 mm. A test piece for attachment measurement was prepared. Using Spotchem SP-4410 (manufactured by Kyoto Daiichi Kagaku Co., Ltd.), sample: KCl aqueous solution (see FIG. 1 for concentration) Sample amount: 7 μl Reaction temperature: 37 ° C. Measurement wavelength: 550 nm from the development layer side The reflectance was measured. The reflectance is K / according to the Kubelka-Munk equation.
The value was converted into an S value and the amount of increase (ΔK / S) for 5 to 10 minutes was determined. The results are shown in Fig. 1. This curve was used as a calibration curve.

Example 2 Using human pool serum as a sample, sample amount 7 μl, reaction temperature 3
Measurement was carried out in the same manner as in Example 1 under the conditions of 7 ° C., measurement wavelength of 550 nm, and measurement time of 5 to 10 minutes, and the obtained ΔK / S was converted into potassium ion concentration by the calibration curve of FIG. A value of 2 mmol / l was obtained. This was in good agreement with the result measured by the ion-selective electrode method (apparatus used: Spotchem SE-1510, manufactured by Kyoto Daiichi Kagaku Co., Ltd.).

Example 3 Using human pool urine as a sample, sample amount 7 μl, reaction temperature 37
Measurement was carried out in the same manner as in Example 1 under the conditions of ℃, measurement wavelength of 550 nm and measurement time of 5 to 10 minutes, and the obtained ΔK / S is shown in FIG.
When converted to potassium ion concentration by the calibration curve of
A value of 28.2 mmol / l was obtained. This is in good agreement with the result measured by the ion selective electrode method (apparatus used:
Spot Chem SE-1510. Made by Kyoto Daiichi Kagaku Co., Ltd.).

Example 4 The effect of sodium ions on the dry test piece for measuring potassium ion concentration according to the present invention was investigated. 0, 10, 20, 30, 40 or 50 sodium chloride in serum
The potassium ion concentration was obtained in the same manner as in Example 1 by adding to a concentration of mmol / l. The results are shown in Figure 2.

Serum contains sodium ions at a concentration of about 140 mmol / l in a healthy person. If the NAD synthase is also activated by sodium ion, the color development does not show a constant of 4.0 mmol / l and the straight line rises to the right as the amount of sodium added increases. Even if saturated with sodium, the ΔK / S value goes on a straight line almost horizontally, but in that case, the coloration corresponding to about 4.0 mmol / l (serum potassium concentration of healthy person) is not shown, but it is darker than that. It should develop color and become impossible to measure. In this example, even if the amount of sodium added was increased, the degree of color development did not change, and the potassium concentration was about 4.0 mmol / l, which was a substantially constant value. From this, it can be seen that NAD synthase is not affected by sodium ions at all, but is affected by potassium alone.

Thus, when NAD synthase is used, potassium ions in the range of at least 0 to 50 mmol / l are
It can be quantified without any dilution procedure or binder. Therefore, it can be incorporated in the same test piece as the test piece of the multi-item test for checking the degree of color development such as glucose and protein.

[Brief description of drawings]

FIG. 1 is a potassium chloride concentration of 0 to 10 in Example 1.
mmol / l calibration curve.

FIG. 2 is a graph showing the effect of sodium addition when measuring potassium ion in human pool serum in Example 4.

Claims (4)

[Claims] 1. A composition for measuring potassium ion in a sample solution, which comprises oxidized nicotinamide adenine dinucleotide synthase [EC 6.3.5.1. ], Adenosine triphosphate, divalent metal ion, deamidonicotinamide adenine dinucleotide, amino group donor, dehydrogenase and its corresponding substrate, tetrazolium salt and diaphorase reagent composition for potassium ion measurement . 2. A support, a reagent layer formed on the support, and a spreading layer formed on the reagent layer, wherein any one of the reagent layer and the spreading layer is oxidized nicotinamide. Adenine dinucleotide synthase [EC 6.3.5.
1. ], Adenosine triphosphate, divalent metal ion, deamidonicotinamide adenine dinucleotide, amino group donor, dehydrogenase and its corresponding substrate, tetrazolium salt, diaphorase, high molecular weight polymer and buffer containing potassium ion Test piece. 3. The test piece for measuring potassium ion according to claim 2, wherein the high molecular polymer contained in the reagent layer and / or the spreading layer is a synthetic high molecular polymer. 4. An oxidized nicotinamide adenine dinucleotide synthase is contained in a reagent layer, and at least one of adenosine triphosphate, a divalent metal ion, deamidonicotinamide adenine dinucleotide and an amino group donor is used.
The test piece for potassium ion measurement according to claim 2, wherein one is included in the spreading layer but not included in the reagent layer.