JP2003274996A - Method of avoiding influence of foreign substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for avoiding influences of foreign substances on measurement results. <P>SOLUTION: When organic components from living bodies are measured through the hydrogen peroxide determining system in which oxidase and peroxidase are conjugated as a detection reaction system, the reagents are constituted from two or more kinds of solutions and the addition order of the solutions are specified. In this case, the first reagent includes catalase and the catalase reaction on the detection reaction is inhibited with an azide of 5-500 mg/L. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for avoiding the influence of contaminants. Specifically, when hydrogen peroxide is produced from the analyte present in the sample and the analyte is detected by measuring the produced hydrogen peroxide,
The present invention relates to a method for inhibiting catalase activity when detecting an analyte after eliminating hydrogen peroxide derived from a substance (interfering substance) other than the analyte using catalase.

[0002]

2. Description of the Related Art When an analyte in a sample such as body fluid is detected using an enzyme, the detection reaction system includes a reaction system for coupling a dehydrogenase with NAD (P) H, which is a coenzyme, and an oxidation reaction. Generally, there are many hydrogen peroxide measurement systems that couple an enzyme with peroxidase. The latter is a method in which an oxidase is allowed to act on an analyte or a reaction product thereof, and hydrogen peroxide produced is converted to a quinoneimine dye, for example, by a peroxidase and a chromogen, and the dye is subjected to absorbance measurement for colorimetric determination. . When detecting an analyte, the oxidase may act directly on the analyte, but in many cases, in addition to the oxidase and peroxidase, 1
Requires more than one type of other enzyme.

[0003] Examples of requiring one or more kinds of other enzymes in addition to the oxidase as an analyte include triglyceride, creatinine, creatine, free cholesterol, cholesterol ester, phospholipid, inorganic phosphorus, amylase, G
Examples include OT, GTP, sialic acid and guanase.

In recent years, in order to improve the accuracy of clinical test reagents, avoiding the influence of contaminants in biological samples has become an important factor in reagent performance. There is a problem that an endogenous substance involved in the above-mentioned enzyme reaction system is present in a sample containing these analytes, for example, body fluid, and a positive error occurs in the measurement of the objective analyte. For example, when measuring triglyceride, when measuring free glycerol and creatinine,
When measuring creatine and sarcosine and ester type cholesterol, when measuring free cholesterol and phospholipid, when measuring choline and inorganic phosphorus, when measuring hypoxanthine and amylase activity, when measuring glucose, GOT or GTP activity Examples include pyruvic acid and sialic acid, pyruvic acid and guanase, and uric acid.

Conventionally, one of the methods that have been carried out as a measure against the problem is a method of subtracting an endogenous contaminant as a sample blank. Although this method can prevent an error, it requires a special reagent as a sample blank, requires additional measurement operation, and is not practical due to its low applicability to automatic analyzers and the like.

[0006] As another method, hydrogen peroxide generated by the action of a specific oxidase on a contaminant is converted into a colorless substance by catalase to eliminate it, and then catalase which is unnecessary for this reaction is removed. Methods of inhibiting activity with inhibitors are known.

For example, in the measurement of creatinine, creatine produced by the action of creatinine amidohydrolase on creatinine also exists in the living body, and therefore, when creatinine is measured, it is necessary to delete creatine in advance. In order to eliminate endogenous creatine, a two-reagent system creatinine measuring reagent using catalase is known (Japanese Patent Publication No. 4-34400).

Examples of creatinine measuring reagents include those having the following composition. Reagent R1: creatine amidinohydrolase, sarcosine oxidase, catalase, reagent R2: creatinine amide hydrolase, peroxidase, chromogen.

That is, in the first enzymatic reaction used for the reagent R1, creatine in the sample is converted into a colorless substance by the reaction of creatine amidinohydrolase, sarcosine oxidase and catalase, and erased.

Then, as a second enzymatic reaction, the reagent R2 was added to the system of the sample and the reagent R1 to react the creatinine in the sample with creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, chromogen and peroxidase. A colored material is produced and the material is colorimetrically determined.

Catalase that coexists in the second enzymatic reaction can be blocked by adding the catalase inhibitor. Similarly to creatinine, elimination of hydrogen peroxide derived from other than the analyte is also required in detection such as triglyceride and inorganic phosphorus measurement.

Sodium azide has heretofore been widely used as the above-mentioned catalase inhibitor.

[0013]

It is known that an azide has an explosive property by forming a metal salt, and it is desirable that its content in the reagent solution is small. Further, it is known that a toxic azide gas is generated in an acidic region during preparation of a test solution, and it is desirable that the content is small. Also, sodium azide is a substance that is not directly involved in the measurement reaction, but since many substances are mixed in biological samples, unknown components in the sample and sodium azide or sodium azide preparations as raw materials There is a possibility of non-specific reaction with impurities in the solution. From this point as well, it is desirable that the content in the reagent solution is low.

[0014]

As a result of various studies to achieve the above object, the present inventors have found that lithium azide has a higher ability to inhibit catalase activity than sodium azide. The inventors have found that the amount of azide in a reagent can be suppressed to a low level by using lithium azide, and have completed the present invention.

That is, the present invention is as follows: (1) When a biological component is measured via a hydrogen peroxide measuring system that couples an oxidase and a peroxidase as a detection reaction system, the reagent is composed of two or more kinds of solutions, and the reaction solution In a measurement method that is specified to be added in a specific order, the first reagent contains catalase,
In the method of inhibiting the catalase reaction during the detection reaction,
A method for avoiding the influence of contaminants, which comprises using 5-500 mg / L of azide. (2) The method according to (1), wherein the azide is at least one azide selected from monovalent alkali metal salts. (3) The method according to (2), wherein the azide is lithium azide. (4) The method according to (1) to (3), wherein the pH of the reagent is 5 to 10. Is.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, "inhibition of catalase activity" means that hydrogen peroxide produced in this reaction is not eliminated by the reaction of catalase.

In the present invention, "contaminant" means free glycerol when measuring ascorbic acid or neutral fat which is a reducing substance, creatine and sarcosine when measuring creatinine, and free cholesterol when measuring ester cholesterol. , Glucose for measuring amylase activity, pyruvic acid for measuring sialic acid, and uric acid for measuring guanase activity.

In the present invention, "oxidizing enzyme" means ascorbate oxidase, glycerol oxidase,
There are glycerophosphate oxidase, sarcosine oxidase, cholesterol oxidase, glucose oxidase, pyruvate oxidase, uricase, etc.,
Any source may be used as long as it is an oxidase that generates hydrogen peroxide.

The catalase used in this invention is
It can be of any origin. Examples include animal-derived substances contained in liver, red blood cells, stomach, etc., and those contained in microorganisms such as Micrococcus sp., Aspergillus sp. Conventionally, many enzymes derived from bovine liver have been used. In addition to the above, it is possible to use catalase derived from other animals or microorganisms.

The catalase activity inhibitor used in the present invention is an azide. The azide may be a compound with a monovalent alkali metal, and examples thereof include sodium azide and lithium azide, but it is preferable to use lithium azide.

The concentration of azide used is 5 to 500 m.
The ability to inhibit catalase activity is recognized in the range of g / dL, but it is preferably 5 to 100 mg / dL, and more preferably 10 to 50 mg / dL.

The buffer solution is not particularly limited and may be of any type as long as it can maintain a pH suitable for the reaction system. Examples of commonly used buffers include Tris buffer, phosphate buffer, GOOD buffer and the like. Among them, the Tris buffer solution and the phosphate buffer solution have the advantage of being inexpensive, although the pH is likely to change depending on the concentration and temperature. On the other hand, the GOOD buffer contains MES and Bis-
Tris, ACES, BES, MOPS, PIPES,
TES, HEPES, Tricine, Bicine,
Examples include POPSO, TAPS, CHES, CAPS and the like.

The pH to be used is not particularly limited, but the pH used for ordinary enzyme reaction is 5-10.
It should be in the range of. It is preferable to use it within a pH range of 6-8.

Next, a specific method for quantifying the substrate or enzyme activity will be described.

After the first reagent is added to the quantitative sample of neutral fat (triglyceride), the second reagent is added to the sample and the resulting absorbance is colorimetrically determined. 1st reagent glycerol kinase glycerol phosphate oxidase catalase adenosine 3 phosphate 4 aminoantipyrine 2nd reagent lipoprotein lipase peroxidase lithium azide DEA (diethylaniline) The reaction system is shown in Formula 1.

[0026]

[Formula 1]

After the addition of the first reagent to the quantitative sample of creatinine, the second reagent is added, and the colorimetric determination is carried out from the resulting absorbance. First reagent creatine amidinohydrase sarcosine oxidase catalase 4 aminoantipyrine 2nd reagent creatinine amididohydrase peroxidase lithium azide DEA (diethylaniline) The reaction system is shown in Formula 2.

[0028]

[Formula 2]

After the first reagent was added to the quantitative sample of ester cholesterol, the second reagent was added to the sample, and colorimetric determination was carried out from the resulting absorbance. First reagent cholesterol oxidase catalase 4 aminoantipyrine Second reagent cholesterol esterase peroxidase lithium azide DEA (diethylaniline) The reaction system is shown in Formula 3.

[0030]

[Formula 3]

After the first reagent is added to the amylase quantitative sample, the second reagent is added to the sample and the resulting absorbance is colorimetrically determined. First Reagent Glucose Oxidase β-Glucosidase Catalase 4 Aminoantipyrine Second Reagent Starch (Substrate) Peroxidase Lithium Azide DEA (Diethylaniline) The reaction system is shown in Formula 4.

[0032]

[Formula 4]

Quantitative determination of sialic acid After the first reagent is added to the sample, the second reagent is added to carry out colorimetric quantification from the resulting absorbance. First reagent pyruvate oxidase catalase 4 aminoantipyrine Second reagent neuraminidase peroxidase lithium azide DEA (diethylaniline) The reaction system is shown in Formula 5.

[0034]

[Formula 5]

After the addition of the first reagent to the quantitative sample of guanase, the second reagent is added to perform the colorimetric quantification based on the resulting absorbance. First Reagent Xanthine Oxidase Uricase Catalase 4 Aminoantipyrine Second Reagent Guanine (Substrate) Peroxidase Lithium Azide DEA (Diethylaniline) The reaction system is shown in Formula 6.

[0036]

[Formula 6]

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples. The present invention is not particularly limited to the examples.

(Example 1) Catalase activity inhibition rate when azide was added to an aqueous solution containing catalase at 5 to 1000 mg / dL (when the activity value without addition of azide was 0%) Relative activity ratio) was examined. The method for measuring catalase activity was performed using the titanium coloration method.

(Reagent preparation) PIPES buffer (pH 7.0) 50 mM Catalase (from beef liver) 1000 U / mL Azide 5-1000 mg / dL

[0040]

[Table 1]

(Results) The results are shown in Table 1. It can be seen that the inhibition rate of catalase activity is significantly higher in lithium azide at all addition concentrations.

[0042]

In the present invention, by adding lithium azide as an azide, the catalase activity can be inhibited with a smaller amount than sodium azide. Therefore, it is possible to reduce the content of the azide, which is a dangerous substance, and it is also possible to reduce the frequency of generation of the azide gas, which is a problem during reagent preparation.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2G045 AA28 BA01 BB44 BB51 DA20                       DA31 DA42 DA43 DA61 DA69                       DA70 DB21 FB01 GC10 GC12                 4B063 QA01 QQ61 QR02 QR03 QS20                       QS36 QX01

Claims (4)

[Claims] 1. When measuring a biological component via a hydrogen peroxide measuring system that couples an oxidase and a peroxidase as a detection reaction system, the reagent is composed of two or more kinds of solutions, and the addition to the reaction solution is specific. Contaminant, characterized by using 5-500 mg / L of azide in the method of assaying to be performed in order, wherein the first reagent contains catalase and the catalase reaction is inhibited during the detection reaction. How to avoid the effects. 2. The method according to claim 1, wherein the azide is at least one azide selected from monovalent alkali metal salts. 3. The azide is lithium azide.
The method described. 4. The pH of the reagent is 5-10.
The method described.