JPH0614799A - Reagent composition for removal of buffering of bilirubin and measurement of biological component by using it - Google Patents

Abstract

PURPOSE:To provide the subject method for measuring a biological component by utilizing an oxidase-peroxidase-color developer system while characteristically avoiding the influence of bilirubin coexisting in the measurement system on the reaction and to provide the subject reagent composition therefor. CONSTITUTION:Based on that hydroxylamine, 2-carboxy-5-hydroxy-gamma-pyrrone or 2-chloromethyl-5-hydroxy-gamma-pyrrone remarkably inhibits the effect of bilirubin oxidase on a color developer but almost dose not inhibit the effect on bilirubin, by using the above substance in combination with bilirubin oxidase, bilirubin is removed so as to avoid the buffering effect of bilirubin.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring a biological component which is characterized by avoiding reaction interference of coexisting bilirubin when measuring a biological component by an oxidase-peroxidase-color former system and a reagent used therefor. It relates to a composition. In recent years, in clinical chemistry analysis, progress in enzymatic analysis methods using an oxidase-peroxidase-coloring reagent system has been remarkable. However, when this measurement system is used, it is likely to be interfered with by various reducing substances, administered drugs, biological dyes, etc. existing in the biological sample. In particular, the interference effect of bilirubin, which is one of the vital pigments, is a particularly serious problem, and at present the basic solution has not yet been made.

[0002]

As a conventional technique, for example, in a method of quantifying glucose by a glucose oxidase-peroxidase-coloring reagent system, a method using a ferrocyanide [Japanese Patent Publication No. 55-25840, Clinical Chemistry] , 26, 227-231 (19
1981)], in a method for quantifying uric acid by a uricase-peroxidase-coloring reagent system, a method using a ferrocyanide ion (JP-A-55-138656), a method for quantifying uric acid by a reaction with picric acid Method for inhibiting the oxidation of bilirubin by ascorbic acid (Patent Document 1)
55-29718), a method for correcting interference by bilirubin using a multi-wavelength photometer, a method of adding an EDTA-iron complex (JP-A-57-71398), bilirubin-specific bacterial enzyme or bilirubin Method of eliminating bilirubin by adding oxidase to the reaction system (Japanese Patent Publication No. 55-25840, Japanese Patent Publication No. 55-138656, Japanese Patent Publication No. 55-29718, Japanese Patent Publication No. 57-71398, JP-A-59-18000). However, neither method was satisfactory.

[0003]

In particular, the method for eliminating bilirubin using the above-mentioned bilirubin oxidase is considered to be useful for the following reasons. In other words, bilirubin oxidase is an enzyme that oxidizes bilirubin to produce biliverdin, and the produced biliverdin is further converted by bilirubin oxidase into a colorless substance, and the reductive property is eliminated. For this reaction,
Since no production of hydrogen peroxide is observed, this is a useful method in the art.

However, bilirubin oxidase is a coloring agent such as 4-aminoantipyrine, phenol, N, N-.
Diethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (hereinafter T
There is a drawback that the blank value is increased because it has some reactivity with (such as OOS).

The present invention provides a technique for improving such a drawback, avoiding or reducing the interference of bilirubin by bilirubin oxidase, and improving the measurement accuracy.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems and achieve the object, the present inventors have found that hydroxylamine, 2-carboxy-5-
Hydroxy-γ-pyrone or 2-chloromethyl-5
It was found that -hydroxy-γ-pyrone strongly inhibits the action of bilirubin oxidase on the color former, but hardly inhibits the action on bilirubin. Then, by using these substances together with bilirubin oxidase in the oxidase-peroxidase-color former system, it is possible to eliminate bilirubin by bilirubin oxidase and avoid the interference effect by bilirubin, and at the same time to the measurement system by bilirubin oxidase. The present invention has been completed by discovering that the influence of can be avoided.

That is, according to the present invention, when the biological components contained in a sample such as serum and urine are measured by the oxidase-peroxidase-color former system, the sample is preliminarily analyzed with at least bilirubin oxidase and hydroxylamine, 2-carboxy-5. -Hydroxy-γ-pyrone or 2-chloromethyl-5-hydroxy-γ-pyrone in the presence of a reagent composition containing one or more kinds and a color former,
It is a method of measuring thereafter and a reagent composition used in the method.

The measurement of the oxidase-peroxidase-color former system in the present invention is a method in which hydrogen peroxide is produced from the component to be quantified and then colorimetrically quantified with peroxidase and a color former. Examples include measurement of biological components using an enzyme. Examples include glucose measurement using glucose oxidase as an oxidase, cholesterol measurement using cholesterol oxidase, galactose measurement using galactose oxidase, and uric acid measurement using uricase.

As the peroxidase used in the present invention, any of plant origin, animal origin and microbial origin can be used.

As the color former, a combination of 4-aminoantipyrine and phenol is mainly used. In addition, in order to increase sensitivity, a combination of 3-methyl-2-benzothiazolinone hydrazone and N, N-dimethylaniline, a combination of 4-aminoantipyrine and p-chlorophenol, a combination of 4-aminoantipyrine and TOOS, 4 -Aminoantipyrine and 3-methyl-N-
(Β-hydroxyethyl) -aniline combination, 4
Many color formers such as a combination of -aminoantipyrine and N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine have come to be used. These can be used even if the combination is changed appropriately.

Further, the bilirubin oxidase used in the present invention is, for example, Myrotheciu.
Examples include enzymes produced by strains belonging to the genus m), the genus Trachyderma or the genus Coprinus.

As the reagent composition used for eliminating bilirubin in the subject, for example, the following compositions can be used.

Bilirubin oxidase 0.1-1.0u / ml,
Hydroxylamine 0.1-2.4 mM and / or 2-carboxy-5-hydroxy-γ-pyrone 2-40 μg / ml and / or 2-chloromethyl-5-hydroxy-γ-pyrone 8-200 μg / ml, color development It contains 4-aminoantipyrine in an amount of 0.3 to 3 mM.

The reaction conditions vary depending on the content of bilirubin, the amount of bilirubin oxidase enzyme, etc.
The reagent composition as described above is added to the subject, preferably
30-40 ℃, 1-10 minutes, pH 6.0 ~ 8.0 to react, then,
Biological components can be measured by various methods using various oxidases.

When reacting with an oxidase, sodium azide is added to completely inhibit the activity of the remaining bilirubin oxidase, so that various biological components can be more accurately measured. Hereinafter, the present invention will be described in detail with reference to experimental examples and examples, but the present invention is not limited thereto.

[0016]

Example 1 1.2 ml of the following reagent A was placed in a quartz cell, and then 5
% Triton X-100 0.02 ml, 20 mg / dl albumin-bound bilirubin 0.1 ml and various concentrations of cholesterol 0.06 ml
Was added and incubated at 37 ° C. for 5 minutes. Then, 2.0 ml of reagent B was added and reacted at 37 ° C., and the absorbance at 555 nm was measured. Similarly, the measurement was performed using a reagent system containing no albumin-bound bilirubin.

Reagent A Sodium cholate 20 mg / ml Bilirubin oxidase 0.25 u / ml Hydroxylamine 0.48 mM 4-Aminoantipyrine 0.9 mM

Reagent B TOOS 1.5 mM peroxidase 10 u / ml Cholesterol oxidase 1.5 u / ml Triton X-100 0.1% sodium azide 50 mM Both were dissolved in 0.1 M phosphate buffer (pH 7.0).

As shown in FIG. 1, the absorbance values were the same regardless of the presence or absence of bilirubin, indicating that the interference due to bilirubin was avoided.

On the other hand, the same measurement was carried out in a system in which the reagent A did not contain biruoxidase and hydroxylamine, and the reagent B did not contain sodium azide. As shown in FIG. 2, it can be seen that this system is strongly affected by bilirubin.

Example 2 In place of hydroxylamine in Example 1 , 10 μg /
The same was done using ml of 2-carboxy-hydroxy-γ-pyrone. The result is shown in FIG. The result shows that the interference of bilirubin is avoided as in the first embodiment.

Example 3 In place of hydroxylamine in Example 1 , 50 μg /
The same was done using 2 ml of 2-chloromethyl-5-hydroxy-γ-pyrone. The result is shown in FIG. The result is similar to that of Example 1, and it can be seen that the interference of bilirubin is avoided.

Example 4 The same procedure as in Example 1 was carried out by using bilirubin oxidase derived from Trachyderma spp. The results were the same as in Example 1.

Example 5 A 0.1 ml sample of the following was placed in a quartz cell and
Then, 1.2 ml of the following reagent A was added, and the mixture was incubated at 37 ° C for 5 minutes. Then, 2.0 ml of the reagent B was added, reacted at 37 ° C. for 5 minutes, and the absorbance at 555 nm was measured. On the other hand, similarly, the measurement was carried out using a reagent system containing no bilirubin oxidase, hydroxylamine and sodium azide.

Glucose solution containing sample albumin-bound bilirubin (20 mg / dl) 15 mg / dl Glucose solution 15 mg / dl

Reagent A 4-Aminoantipyrine 0.9 mM Sodium cholate 20 mg / ml Hydroxylamine 0.48 mM Bilirubin oxidase 0.25 u / ml (Mirocesium origin) Peroxidase 10 u / ml

Reagent B TOOS 1.5 mM sodium azide 50 mM glucose oxidase 30 u / ml mutarotase 1 u / ml All were dissolved in 0.1 M phosphate buffer (pH 7.0). The results are shown in Table 1.

[0028]

[Table 1]

It can be seen that the interference of bilirubin was avoided by the reagent system containing bilirubin oxidase, hydroxylamine and sodium azide.

Experimental Example 1 Reagent A described in Example 1 was allowed to stand at 37 ° C. for 1 , 6, 24 and 30 hours and then measured in the same manner as in Example 1 . The cholesterol concentration of the sample was 100 mg / dl.

The same measurement was carried out using 2-carboxy-5-hydroxy-γ-pyrone or 2-chloromethyl-5-hydroxy-γ-pyrone instead of hydroxylamine. As a control, Reagent A containing no such inhibitor was also treated and measured in the same manner. The results are shown in Table 2.

[0032]

[Table 2]

As is clear from Table 2, when reagent A to which an inhibitor such as hydroxylamine has not been added is allowed to stand at 37 ° C. and then cholesterol is determined using reagent B, an accurate measurement value can be obtained. I know there isn't. That is, when the measured value at the time of reagent preparation is 100%, the measured value becomes 70% when the reagent left for 30 hours is used, and the reagent is unstable. Further, the reagent A is obviously browned.

In comparison therewith, hydroxylamine, 2-
Carboxy-5-hydroxy-γ-pyrone or 2-
By containing chloromethyl-5-hydroxy-γ-pyrone, the storage stability of the reagent A can be achieved and accurate measurement can be performed.

Experimental Example 2 The inhibitory effect of hydroxylamine on bilirubin oxidase was examined as follows. When bilirubin is used as a substrate 0.0075u / ml bilirubin oxidase 0.1 ml in a quartz cell
And 0.1 ml of various concentrations of hydroxylamine, 37
Incubated at 0 ° C for 10 minutes. Then 20 μg / ml
2.5 ml of bilirubin of was added and reacted at 37 ° C., and the absorbance at 440 nm was measured. Similarly, water was used instead of hydroxylamine for measurement.

When 4-aminoantipyrine-phenol was used as a substrate To a quartz cell, 0.1 ml of 3 u / ml bilirubin oxidase and 0.1 ml of various concentrations of hydroxylamine were added, and the mixture was incubated at 37 ° C. for 10 minutes.
Incubated for minutes. Then 2.5mM 4-aminoantipyrine, a reagent containing 0.15% phenol 2.5mM
l was added and reacted at 37 ° C., and the absorbance at 505 nm was measured.
Similarly, water was used instead of hydroxylamine for measurement.

When 4-aminoantipyrine-TOOS was used as a substrate: 0.15 ml of 0.75 u / ml bilirubin oxidase was added to a quartz cell.
Add 0.1 ml of various concentrations of hydroxylamine, 37 ℃, 1
Incubated for 0 minutes. Thereafter, 2.5 ml of a reagent containing 0.3 mM 4-aminoantipyrine and 1.0 mM TOOS was added, the mixture was reacted at 37 ° C., and the absorbance at 555 nm was measured. Similarly, water was used instead of hydroxylamine for measurement.
All reagents of ~ were dissolved using 0.1 M phosphate buffer (pH 7.0). The results are shown in Table 3.

[0038]

[Table 3]

Experimental Example 3 Regarding 2-carboxy-5-hydroxy-γ-pyrone
It carried out like Experimental example 2 . The results are shown in Table 4.

[0040]

[Table 4]

Experimental Example 4 The same procedure as in Experimental Example 2 was repeated for 2-chloromethyl-5-hydroxy-γ-pyrone. The results are shown in Table 5.

[0042]

[Table 5]

From Experimental Examples 2 to 4 , hydroxylamine,
Both 2-carboxy-5-hydroxy-γ-pyrone and 2-chloromethyl-5-hydroxy-γ-pyrone were treated with 4-aminoantipyrine / phenol and 4-aminoantipyrine / TOOS in comparison with the case where bilirubin was used as a substrate. The substrate was more effective in inhibiting the activity of bilirubin oxidase. Especially when hydroxylamine was used, the inhibitory effect was remarkable.

[0044]

According to the present invention, hydroxylamine, 2
-Carboxy-5-hydroxy-γ-pyrone or 2
-Chloromethyl-5-hydroxy-γ-pyrone inhibits the reaction of bilirubin oxidase to 4-aminoantipyrine, phenol, N, N-diethylaniline, TOOS, etc., but hardly inhibits the action on bilirubin. Then, by allowing this substance to coexist with bilirubin oxidase in the measurement of oxidase-peroxidase-chromogen system, it is possible to eliminate bilirubin by bilirubin oxidase and avoid the interference effect, and at the same time, to the measurement system using bilirubin oxidase. The influence can be avoided.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 1, in which circles show results when albumin-bound bilirubin was not included, and triangles show results when albumin-bound bilirubin was included.

FIG. 2 is a graph showing the measurement results in the case of not containing bilirubin oxidase, hydroxylamine and sodium azide in Example 1, in which the circles represent the results when albumin-bound bilirubin was not contained and the triangles represent albumin. The results when the bound bilirubin is included are shown.

FIG. 3 is a graph showing the results of Example 2, in which the circles show the results when albumin-bound bilirubin was not included, and the triangles show the results when albumin-bound bilirubin was included.

FIG. 4 is a graph showing the results of Example 3, in which the circles show the results when albumin-bound bilirubin was not included, and the triangles show the results when albumin-bound bilirubin was included.

Claims (2)

[Claims] 1. A reagent composition for measuring a biological component using an oxidase, a peroxidase and a color former, which comprises at least bilirubin oxidase and hydroxylamine.
-Carboxy-5-hydroxy-γ-pyrone or 2
-A reagent composition for removing interference of bilirubin, which comprises one or more of chloromethyl-5-hydroxy-γ-pyrone and a color former. 2. In the measurement of biological components using oxidase, peroxidase and color former, at least bilirubin oxidase and hydroxylamine, 2-carboxy-5-hydroxy-γ-pyrone or 2-chloromethyl-5-hydroxy are previously prepared. A method of removing interference of bilirubin by treating with a reagent composition containing one or more γ-pyrone and a color former.