JP3522307B2 - Liquid reagent composition for measuring inorganic phosphorus - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid reagent composition for measuring inorganic phosphorus in a sample, particularly blood and urine, and a method for measuring inorganic phosphorus.

[0002]

2. Description of the Related Art Conventionally, the molybdic acid method has been used as a method for measuring inorganic phosphorus in blood and urine. However, this method has various problems such as complicated operation, low specificity, corrosion of equipment and coloring. In order to avoid these problems, a method for measuring inorganic phosphorus by an enzymatic method has been developed in recent years and is widely used because of its superiority in precision and convenience. The main enzymatic method is purine nucleoside phosphorylase-
A system using xanthine oxidase is known.

On the other hand, recently, in the in-vitro diagnostic agent using the enzyme method, stabilization of the enzyme as a raw material, screening of thermostable enzyme,
What was conventionally distributed as a freeze-dried product due to modification of enzyme properties by protein engineering techniques has improved reagent stability due to improved stability, purity, etc., and is now distributed in liquid form. There is. However, it cannot be said that the problems of stability and purity of raw materials have been solved yet, and it is the current situation that the raw materials are commercialized by carefully selecting raw materials and studying a combination of raw materials having good reagent stability.

[0004]

The determination of inorganic phosphorus is no exception, and it is not affected by interfering substances in the sample such as ascorbic acid or endogenous xanthine, hypoxanthine or endogenous inosine in the sample, and long-term storage is possible. Development of liquid reagent compositions with excellent stability and compatibility with automatic analyzers is in progress. A conventionally known reagent for measuring inorganic phosphorus has the following composition. Ascorbate oxidase,
It consists of a first reagent solution containing xanthine oxidase, peroxidase and chromogen, and a second reagent solution containing xanthine oxidase, 4-aminoantipyrine, inosine and purine nucleoside phosphorylase. This reagent has a problem that the reagent blank increases during long-term storage.

In order to solve the above problems, a first reagent solution containing ascorbate oxidase, xanthine oxidase, purine nucleodophosphorylase and a chromogen, xanthine oxidase, 4-aminoantipyrine,
A reagent for measuring inorganic phosphorus, which comprises a second reagent solution containing inosine and peroxidase, is known. However, in the case of such a two-reagent system, when purine nucleoside phosphorylase is contained in the first reagent solution, inorganic phosphorus is consumed unnecessarily, which has a negative effect. Therefore, it is difficult to simultaneously solve the problem that the reagent blank increases during long-term storage and the problem that it is affected by inosine in the sample. Therefore, by solving both of the above two problems,
Moreover, there is no deterioration of the reagent, and there is a need for a liquid reagent composition that can accurately measure inorganic phosphorus without being affected by interfering substances in the sample even after long-term storage, endogenous xanthine, hypoxanthine, inosine, etc. .

In view of the above situation, an object of the present invention is that it is convenient for normal use, and interferes with an analyte, endogenous xanthine,
It is an object of the present invention to provide a liquid reagent composition for measuring inorganic phosphorus, which is not affected by hypoxanthine and inosine, can retain reagent performance even during long-term storage, and has excellent compatibility with an automatic analyzer.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to achieve the above object. As a result, purine nucleoside phosphorylase, xanthine oxidase, and inosine are the main components, and hydrogen peroxide produced is hydrogen peroxide. In the presence of a substance having a resolving power of 2, a liquid reagent composition consisting of 2 reagents for detecting inorganic phosphorus by reacting with a chromogen and a coupler and measuring the optical absorbance,
Purine nucleoside phosphorylase is added only to the second reagent solution, xanthine oxidase is added to both the first and second reagent solutions, and inosine is contained in the second reagent solution, and by containing catalase in the second reagent solution, It was found that the interference substances in the sample, endogenous xanthine, hypoxanthine, and inosine are not affected, the reagent performance is maintained even during long-term storage, it is compatible with an automatic analyzer, and inorganic phosphorus is accurately measured, Was completed.

That is, according to the present invention, the first reagent solution contains xanthine oxidase, does not contain purine nucleoside phosphorylase, and the second reagent solution is xanthine oxidase.
A liquid reagent composition for measuring inorganic phosphorus, comprising a purine nucleoside phosphorylase, inosine and catalase, and a first reagent solution and / or a second reagent solution containing a peroxidase, a chromogen and a coupler.

In the present invention, the first reagent solution containing xanthine oxidase is added to a sample to eliminate endogenous xanthine and hypoxanthine in the sample, and then xanthine oxidase, purine nucleoside phosphorylase, inosine and catalase are added. A method for measuring inorganic phosphorus, characterized in that after adding the second reagent solution contained therein, the produced hydrogen peroxide is reacted with a chromogen and a coupler in the presence of peroxidase, and the generated optical absorbance is measured. .

The purine nucleoside phosphorylase [EC 2.4.2.1] used in the present invention acts on inorganic phosphorus and inosine to form hypoxanthine and ribose-1.
-It is an enzyme that produces phosphate, and its origin is not particularly limited. Derived from animal organs, such as Human erythrocytes,
Calf spleen etc. are mentioned. Microbes, such as Enteroba
Examples include those derived from cter cloacae and Bacillus cerus.

The xanthine oxidase [EC 1.1.3.22] used in the present invention is an enzyme which acts on endogenous xanthine or hypoxanthine in the presence of oxygen to produce uric acid and hydrogen peroxide. The origin is not particularly limited. Milk-derived or microorganisms such as Arth
Those derived from robacter luteus are mentioned.

The chromogen used in the present invention is a substance which develops a color by reacting with the following coupler in the presence of hydrogen peroxide and a substance capable of decomposing hydrogen peroxide.
Ethyl-N- (3-sulfopropyl) -m-anisidine, N-ethyl-N- (3-methylphenyl) -N'-
Succinylethylenediamine, N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-)
Examples thereof include aniline derivatives such as sulfopropyl) -m-toluidine and N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline.

The coupler used in the present invention is a substance that develops a color by combining with the above chromogen in the presence of hydrogen peroxide and a substance capable of decomposing hydrogen peroxide, such as 4-aminoantipyrine and 3-methyl. -2-benzothiazoline hydrazine etc. are mentioned.

Examples of the substance capable of decomposing hydrogen peroxide used in the present invention include peroxidase and catalase.

In order to eliminate interfering substances such as ascorbic acid in the specimen, ascorbate oxidase is first added.
It is preferably contained in the reagent.

As a specific example of the reagent of the present invention, an inorganic phosphorus consisting of a first reagent solution containing xanthine oxidase, peroxidase and chromogen and a second reagent solution containing xanthine oxidase, coupler, inosine, purine nucleoside phosphorylase and catalase. There is a liquid reagent composition for measurement. The first reagent solution of the present invention optionally contains ascorbate oxidase, but does not contain purine nucleoside phosphorylase.

The second reagent solution of the present invention contains xanthine oxidase, a coupler, inosine, purine nucleotide phosphorylase and catalase.

In addition to the above components, the first reagent solution and the second reagent solution contain a buffer and, if necessary, a surfactant, a stabilizer,
Contains preservatives and reagents to remove interfering substances. As the buffering agent, an ordinary one is used, and it is preferable to adjust the pH of the first reagent solution and the second reagent solution to 5 to 11.

The purine nucleoside phosphorylase used in the present invention has an enzyme concentration of 0.2 to 5 in the second reagent solution.
It is preferably used in the range of U / ml. The enzyme concentration of xanthine oxidase is 0.5 to 20 U in the first test solution.
/ Ml, and preferably in the range of 0.01 to 5 U / ml in the second reagent solution. The concentration of the chromogen used in the present invention is preferably in the range of 1 × 10 ⁻⁶ to 1 × 10 ⁻² M in the final reaction solution. The concentration of the coupler used in the present invention is preferably in the range of 1 × 10 ⁻⁷ to 1 × 10 ⁻² M in the final reaction solution. The concentration of catalase used in the present invention is preferably in the range of 0.1 to 10 U / ml. If the concentration of peroxidase is suitable for measurement,
It is not particularly limited.

The following reaction is carried out in order to measure the inorganic phosphorus in a sample using the liquid reagent composition comprising the two liquids of the present invention.

(First stage reaction) Elimination of endogenous xanthine or hypoxanthine

[Chemical 1]

[0022]

[Chemical 2]

Elimination of endogenous ascorbic acid

[Chemical 3]

(Second stage reaction) Measurement of inorganic phosphorus

[Chemical 4]

[0025]

[Chemical 5]

[0026]

[Chemical 6]

In the present invention, the purine nucleoside phosphorylase is contained only in the second reagent solution, so that when measuring a sample in which inosine is present, unnecessary consumption of inorganic phosphorus is eliminated in the first reagent solution to reduce an error in the measured value. Further, by containing inosine, xanthine oxidase and catalase in the second reagent solution, it is possible to prevent the reagent blank from rising. In addition, organic phosphorus is present in the reagent components, and these may become inorganic phosphorus during long-term storage due to the action of phosphatase or spontaneous decomposition. This phenomenon causes the reagent blank to rise during long-term storage of the reagent, impairing the reagent performance. In addition, xanthine oxidase significantly impairs long-term storage stability due to coexistence with inosine. Due to this phenomenon, when inosine and xanthine oxidase are present in the first reagent solution, the ability to eliminate xanthine and hypoxanthine is lost, and the reagent performance is impaired. Therefore, purine nucleoside phosphorylase,
By having inosine, xanthine oxidase, and substances that decompose hydrogen peroxide, excellent long-term storage stability,
It has been possible to complete a reagent composition for measuring inorganic phosphorus that is not affected by interfering substances, endogenous xanthine, hypoxanthine, and inosine in a sample and is excellent in precision and convenience.

Next, the invention will be described in detail with reference to Examples. Example 1 Reagents A to E having the following compositions were prepared, and immediately after preparation, at 4 ° C.
It was stored at -10 ° C for 7 months and 13 months. The concentration of inorganic phosphorus in the test sample was measured by the following method using each reagent after storage. Reagent A 1st reagent Good buffer solution (pH 6.8) 50 mM Ascorbate oxidase 2 U / ml Xanthine oxidase 5 U / ml Peroxidase 10 U / ml N-Ethyl-N- (3-sulfopropyl) -m-anisidine 2 mM 2nd reagent Good Buffer solution (pH 6.8) 50 mM xanthine oxidase 2 U / ml 4-aminoantipyrine 1 mM inosine 37 mM purine nucleoside phosphorylase 1 U / ml catalase 10000 U / ml

[0029] Reagent B (comparative example)   First reagent     Good buffer solution (pH 6.8) 50 mM     Ascorbate oxidase 2U / ml     Xanthine oxidase 5U / ml     Peroxidase 10U / ml     N-ethyl-N- (3-sulfopropyl) -m-anisidine 2 mM     Inosine 24.7 mM   Second reagent     Good buffer solution (pH 6.8) 50 mM     Xanthine oxidase 2U / ml     4-aminoantipyrine 1 mM     Purine nucleoside phosphorylase 1U / ml     Catalase 10U / ml

[0030] Reagent C (comparative example)   First reagent     Good buffer solution (pH 6.8) 50 mM     Ascorbate oxidase 2U / ml     Xanthine oxidase 5U / ml     Peroxidase 10U / ml     N-ethyl-N- (3-sulfopropyl) -m-anisidine 2 mM     Purine nucleoside phosphorylase 0.67U / ml   Second reagent     Good buffer solution (pH 6.8) 50 mM     Xanthine oxidase 2U / ml     4-aminoantipyrine 1 mM     Inosine 37 mM     Catalase 10U / ml

[0031] Reagent D (Comparative example)   First reagent     Good buffer solution (pH 6.8) 50 mM     Ascorbate oxidase 2U / ml     Xanthine oxidase 5U / ml     Peroxidase 10U / ml     N-ethyl-N- (3-sulfopropyl) -m-anisidine 2 mM   Second reagent     Good buffer solution (pH 6.8) 50 mM     Xanthine oxidase 2U / ml     4-aminoantipyrine 1 mM     Inosine 37 mM     Purine nucleoside phosphorylase 1U / ml

[0032] Reagent E (comparative example)   First reagent     Good buffer solution (pH 6.8) 50 mM     Ascorbate oxidase 2U / ml     Xanthine oxidase 5U / ml     Peroxidase 10U / ml     N-ethyl-N- (3-sulfopropyl) -m-anisidine 2 mM   Second reagent     Good buffer solution (pH 6.8) 50 mM     4-aminoantipyrine 1 mM     Inosine 37 mM     Purine nucleoside phosphorylase 1U / ml     Catalase 10000U / ml

Measurement method Using distilled water and standard (containing 5.0 mg / dl of inorganic phosphorus) as samples, 30 μl of each was sampled, 2.6 ml of the first reagent solution was added thereto, and the mixture was reacted at 37 ° C. for 5 minutes. ,
Add 1.3 ml of the second reagent solution and react at 37 ° C for 5 minutes,
A chromogenic substance of 4-aminoantipyrine and N-ethyl-N- (3-sulfopropyl) -m-anisidine was measured at a wavelength of 600.
It was measured in nm. The results are shown in Table 1 and FIG.

[0034]

[Table 1]

Example 2 Using Reagents A to E of Example 1, 1) Inosine was added to normal serum as a sample at 0, 6, 12, 18, 24, 30 mg / d.
It was measured according to the measuring method of Example 1 using the one added so as to be 1) and the one added with hypoxanthine to the normal serum so as to be 0, 6, 12, 18, 24, 30 mg / dl. The results are shown in Tables 2 and 3 and FIGS.

[0036]

[Table 2]

[0037]

[Table 3]

From the above results, the reagent A has a lower reagent blank than the reagents D and E, and the rise of the reagent blank is small even after long-term storage. Further, it is understood that the reagent A is superior to the reagents B and C without being affected by hypoxanthine and inosine in the sample.

[0039]

EFFECTS OF THE INVENTION By using the reagent composition of the present invention, it is possible to accurately and easily quantify inorganic phosphorus in a short time without being affected by interfering substances, endogenous xanthine, hypoxanthine and inosine in a sample. Moreover, the reagent composition of the present invention can be stored in a liquid state for a long period of time.

[Brief description of drawings]

FIG. 1 is a diagram showing changes in a reagent blank during long-term storage.

FIG. 2 is a graph showing the influence of inosine concentration on measured values.

FIG. 3 is a graph showing the effect of hypoxanthine concentration on measured values.

Front page continuation (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12Q 1/00-1/70

Claims (3)

(57) [Claims] 1. The first reagent solution contains xanthine oxidase and does not contain purine nucleoside phosphorylase,
The second reagent solution contains xanthine oxidase, purine nucleoside phosphorylase, inosine and catalase, and the first reagent solution and / or the second reagent solution contains peroxidase, a chromogen and a coupler. object. 2. A liquid reagent composition for measuring inorganic phosphorus according to claim 1, which comprises a first reagent solution containing xanthine oxidase, peroxidase and a chromogen and a second reagent solution containing xanthine oxidase, a coupler, purine nucleoside phosphorylase and catalase, and inosine. object. 3. The first reagent solution according to claim 1 is added to a sample to eliminate endogenous xanthine and hypoxanthine in the sample, and then the second reagent solution according to claim 1 is added. Thereafter, the produced hydrogen peroxide is reacted with a chromogen and a coupler in the presence of peroxidase, and the generated optical absorbance is measured, and the inorganic phosphorus measuring method is characterized.