JP2003230400A - Reagent composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a reagent composition which simultaneously comprises a color-developing system hydrogen donor and albumin and can significantly inhibit natural color development during long period storage. <P>SOLUTION: This reagent composition which simultaneously comprises at least a hydrogen donor and albumin and is used for reacting hydrogen peroxide with a hydrogen donor and a hydrogen acceptor to form a coloring matter, and then determining the formed coloring matter is characterized by using an aniline derivative not bound with an alkoxyl group at any of 3- and 5- positions as the hydrogen donor. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the formation of a dye by the reaction of hydrogen peroxide, a hydrogen donor and a hydrogen acceptor,
It relates to a reagent composition for quantifying the formed dye.
The reagent composition of the present invention is useful as a reagent for quantifying cholesterol, especially cholesterol in low-density lipoprotein.

[0002]

2. Description of the Related Art Low-density lipoprotein (LDL) is a major player in cholesterol transport in blood, and an increase in LDL is known to be one of the risk factors for arteriosclerotic diseases. It is clinically extremely useful to simply and separately quantify cholesterol in LDL (hereinafter sometimes referred to as “LDL cholesterol”).

Conventional methods for quantifying LDL cholesterol include a method of determining from two steps of fractionation operation and quantification of cholesterol, total cholesterol, HDL cholesterol,
There is a calculation method based on the Friedewald formula obtained from the triglyceride value.

[0004] Fractionation operations include ultracentrifugation, precipitation, and immunization, but the operation of centrifuging or filtering the sample is required, and there is a problem in simplicity and economy.
In addition, the calculation method by the Friedewald formula is also limited in its use, and there is a problem in accuracy because it does not take individual differences into consideration.

The applicant of the present invention has previously proposed an LD which does not require a fractionation operation.
L-cholesterol quantification method (JP 2001-22439 A)
7) was developed, and this method is currently applied in the clinical laboratory. This method uses the LDL in the sample in the first step.
Other than the above, cholesterol in lipoproteins is selectively eliminated in the presence of abulmin, and LDL cholesterol is quantified in the second step. According to the present quantification method, even when the test sample contains a high concentration of triglyceride (TG) by performing the first step in the presence of albumin,
An accurate quantification can be performed while avoiding the influence. Cholesterol is quantified by forming a pigment by the reaction of hydrogen peroxide, which is generated by the action of cholesterol with cholesterol oxidase, a hydrogen donor, and a hydrogen acceptor, and quantifying the formed pigment. Be seen. Then, the reagent composition used in the first step contains a hydrogen donor and albumin at the same time.

[0006]

However, the reagent composition used in the first step in this quantification method, which simultaneously contains a color-forming hydrogen donor and albumin, gradually develops spontaneous color during long-term storage. There was a problem. This color development affects the reaction absorbance depending on the measurement wavelength used. It was also found that this coloring consumes a part of the components in the first step, and a sufficient reaction with the test sample cannot be obtained. Therefore, avoiding this problem enhances the usefulness as a measurement reagent, and there has been a situation where the method is desired. In addition, this problem is
It is also possible to solve the problem by including the hydrogen donor and albumin in separate reagent compositions, but if this is done, one more reagent composition will be mixed at the time of use, and the operation will become complicated and human error will occur. It is not preferable because it is easy to get up. Therefore, it is desired to solve the above problems in a reagent composition containing a hydrogen donor and albumin at the same time.

Therefore, an object of the present invention is to provide a reagent composition containing a color-forming hydrogen donor and albumin at the same time,
An object of the present invention is to provide a reagent composition that can significantly suppress spontaneous color development during long-term storage.

[0008]

As a result of investigating the cause of color development of a reagent composition, the present inventors have found that the coexistence of albumin and an aniline derivative which is a hydrogen donor causes color development due to dissolved oxygen in the reaction solution. I found out that. Specifically, the aniline derivative is gradually oxidized by dissolved oxygen existing in the reaction solution, and changes into an oxide in which oxygen is added to the 4-position. This reaction is likely to occur in the aniline derivative having an alkoxyl group at the 3- and / or 5-position, and the oxidation increases the reactivity at the 2- or 6-position. Then, a substance derived from abulmine binds to this portion to cause color development.

Then, as a result of studying a method for suppressing the color development, the color development during storage of the reaction solution can be suppressed by using albumin in combination with an aniline derivative having no alkoxyl group bonded at the 3- and 5-positions. ,
As a result, the influence on the LDL cholesterol measurement value was avoided, and it was experimentally confirmed that the reagent composition can be stabilized for a long period of time, and the present invention was completed.

That is, the present invention provides a reagent composition for forming a dye by the reaction of hydrogen peroxide, a hydrogen donor, and a hydrogen acceptor, and quantifying the formed dye, which comprises at least the above hydrogen. A reagent composition comprising a donor and albumin at the same time, wherein the hydrogen donor is an aniline derivative in which an alkoxyl group is not bonded to both the 3-position and the 5-position. To do.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The coloring system hydrogen donor contained in the reagent composition of the present invention is an aniline derivative in which an alkoxyl group is not bonded to both the 3-position and the 5-position, and is a hydrogen acceptor. A dye is formed by a reaction with (coupling reaction) with. Any aniline derivative may be used as long as the alkoxyl group is not bonded to both the 3-position and the 5-position and the dye can be formed by the coupling reaction with the hydrogen acceptor. You can Preferred aniline derivatives used in the composition of the present invention include those having a structure represented by the following general formula (I).

[0012]

[Chemical 2] (However, R ¹ and R ² each independently represent a hydrogen atom or an alkyl group, R ³ represents a sulfoalkyl group or a hydroxysulfoalkyl group, and R ⁴ represents a hydrogen atom or an alkyl group.)

In the general formula (I), when R ¹ and R ² are alkyl groups, the alkyl groups may be linear or branched, and the number of carbon atoms is not particularly limited, but is about 1-5. Is preferable, and about 1 to 2 is particularly preferable. The alkyl moiety in the sulfoalkyl group or the hydroxysulfoalkyl group of R ³ may also be linear or branched, and the number of carbon atoms is not particularly limited, but is preferably about 1-8,
Particularly, about 2 to 4 is preferable. Further, the alkyl group of R ⁴ may be linear or branched, and the number of carbon atoms is not particularly limited, but is preferably about 1 to 6, and particularly 1 to
About 3 is preferable. Among the aniline derivatives represented by the general formula (I), particularly preferred are those having an N-sulfopropylaniline derivative having a structure represented by the following general formula (II) and a structure represented by the following general formula (III). Mention may be made of N-hydroxysulfopropylaniline derivatives.

[0014]

[Chemical 3]

[0015]

[Chemical 4] (However, in the general formulas (II) and (III), R ¹ , R ² and R
⁴ is synonymous with R ¹ , R ² and R ^{4 in the} general formula (I), respectively.

The concentration of the aniline derivative in the reagent composition is
Although not particularly limited, it is usually about 0.5 to 4.0 mmol / L, and more preferably about 1.0 to 3.0 mmol / L, based on the weight of the entire reagent composition.

The albumin contained in the reagent composition of the present invention is not limited as long as it is albumin,
Commercially available albumin such as serum albumin can be used. The origin of albumin is not particularly limited, and bovine serum albumin which is widely used can be preferably used.

Although the concentration of albumin in the reagent composition is not particularly limited, it is usually preferably about 0.3 to 3.0%, more preferably 0.5 to 3.0%, based on the total weight of the reagent composition. It is about 1.5%.

Oxidizing enzyme is caused to act on the compound to be measured contained in the test sample, and a dye is formed by the reaction of hydrogen peroxide generated thereby, a hydrogen donor and a hydrogen acceptor, and the formed dye. A one-reagent system or two-reagent system reagent composition for colorimetrically quantifying TG is well known, and is a reagent composition used in a method for selectively quantifying LDL cholesterol, wherein TG is highly concentrated in a test sample. In order to avoid a measurement error that occurs when it is contained in the reagent composition, a reagent composition further containing abulmine is known (Japanese Patent Laid-Open No. 2001-2243).
97). The reagent composition of the present invention is characterized by using the specific aniline derivative described above as a hydrogen donor in a reagent composition containing albumin and a chromogenic hydrogen donor, and other components are known reagents. It may be similar to the composition.

The reagent composition of the present invention may be applied to any application as long as it contains albumin and the above aniline derivative.
It is particularly suitable for the reagent composition used in the first step of the method for quantifying LDL cholesterol described in 224397. Hereinafter, the method for quantifying LDL cholesterol will be further described.

Cholesterol contained in lipoprotein includes ester cholesterol (cholesterol ester) and free cholesterol. In the present specification, the term “cholesterol” simply includes both of them.

The test sample used in this method is HD
Any sample may be used as long as it may contain lipoproteins such as L, LDL, VLDL and CM.
Examples thereof include, but are not limited to, body fluids such as blood, serum and plasma, and dilutions thereof.

The method comprises a first step and a second step, in which the HDL, VLDL and C in the sample to be tested are contained.
The cholesterol in M is eliminated, and in the subsequent second step, the residual cholesterol in the test sample is quantified. Since cholesterol in HDL, VLDL and CM is eliminated in the first step, cholesterol quantified in the second step is mainly cholesterol in LDL in the test sample.

"Elimination" in the first step means that cholesterol is decomposed and its decomposition product is not detected in the next second step. The following method can be mentioned as a method for selectively eliminating cholesterol contained in lipoproteins other than LDL, that is, HDL, VLDL, CM and the like.

That is, cholesterol esterase and cholesterol oxidase are allowed to act in the presence of a surfactant that acts on lipoproteins other than low-density lipoprotein to eliminate hydrogen peroxide produced.

Examples of the method for eliminating hydrogen peroxide include a method in which catalase is caused to decompose into water and oxygen, and a method in which a hydrogen donor is reacted with hydrogen peroxide using peroxidase to convert to colorless quinone. However, the present invention is not limited to these.

The cholesterol esterase concentration in the reaction solution of the first step is preferably about 0.2 to 1.0 U / mL, and the cholesterol oxidase concentration is 0.1 to 1.0 U / mL.
About 0.7 U / mL is preferable. Further, the concentration of catalase is preferably about 400 to 2000 U / mL. The concentration of peroxidase when converting hydrogen peroxide to colorless quinone is preferably 1.0 to 6.0 U / mL,
The concentration of the hydrogen donor is 0.5 to 4.0 mmol / L
Is preferred.

HLB is a preferred example of the surfactant used in the first step, which acts on lipoproteins other than LDL.
Examples thereof include polyalkylene oxide derivatives having a value of 13 or more and 15 or less, preferably 13 or more and 14 or less.

Preferred specific examples of the polyalkylene oxide derivative having an HLB value of 13 or more and 15 or less include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether and polyoxyethylene. Compounds having an HLB value of 13 or more and 15 or less, such as octyl phenyl ether and polyoxyethylene nonyl phenyl ether, can be mentioned, but not limited thereto.

The concentration of the surfactant used in the first step is preferably about 0.1 to 10 g / L, more preferably about 0.5 to 5.0 g / L.

The first step is preferably carried out in a buffer solution having a pH of 5 to 9, and the buffer solution is preferably a buffer solution containing an amine such as Tris, triethanolamine, and Good's buffer solution. In particular, Bis-Tris, P which is a Good's buffer
IPES, MOPSO, BES, HEPES and POP
SO is preferable, and the concentration of the buffer is 10 to 500 mmol
/ L is preferable.

In the first step, a divalent metal ion may be contained in the reaction solution in order to suppress the reaction with LDL and further enhance the elimination of other lipoproteins. Copper ions, iron ions and magnesium ions can be used as the divalent metal ions, and magnesium ions are particularly preferable. The concentration of divalent metal ions is 5 to 200 mmol / L
A degree is preferable.

Incidentally, a lipoproteolytic enzyme can be optionally added to the reaction solution of the first step. The addition of this enzyme is preferable because cholesterol in VLDL easily reacts. The concentration of this enzyme in the reaction solution is preferably about 5.0 to 10.0 U / mL.

The reaction temperature in the first step is preferably about 30 to 40 ° C, and most preferably 37 ° C. The reaction time may be about 2 to 10 minutes.

In the subsequent second step, the residual cholesterol in the test sample is quantified. This is, for example, at least L
It can be carried out by adding a surfactant acting on DL and quantifying the hydrogen peroxide generated by the action of the cholesterol esterase and cholesterol oxidase added in the first step.

Here, at least the surfactant acting on LDL may be a surfactant acting selectively on LDL, or a surfactant acting on all lipoproteins. Preferred examples of the surfactant that acts on all lipoproteins include polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13, preferably 12 or more and less than 13.

Anionic surfactants can be mentioned as the surfactants which selectively act only on LDL. The anionic surfactant used here is not particularly limited, but those having an aromatic ring to which a linear or branched alkyl group having 4 to 18 carbon atoms is bonded are preferable.

The concentration of the surfactant used in the second step is preferably about 0.1 to 100 g / L, more preferably about 1 to 50 g / L.

The quantification of hydrogen peroxide in the second step can be carried out by quantifying the dye formed by the coupling reaction between the hydrogen donor and the hydrogen acceptor. As the hydrogen donor, the above-mentioned aniline derivative (included in the reagent used in the first step) is used, and as the hydrogen acceptor, 4-aminoantipyrine or methylbenzothiazolonehydrazone, etc. is used as in the conventional case. You can

Other preferable conditions for the second step are the first
It is the same as the preferable conditions in the process.

Therefore, when the reagent composition of the present invention is used as a reagent composition for carrying out the first step of the method for quantifying LDL cholesterol described above, cholesterol oxidase, in addition to the above-mentioned albumin and aniline derivative, It may contain at least a surfactant acting on lipoproteins other than cholesterol esterase and LDL, and may further contain catalase, a magnesium salt and a buffer. The concentrations of these components in the composition are preferably set so that the concentrations of the components in the reaction liquid in the first step fall within the above range.

In the method for quantifying LDL, the hydrogen acceptor, which forms a dye by the coupling reaction with the aniline derivative, is added in the second step and is added to the reagent composition used in the first step. Not included.

[0043]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1, Comparative Example 1 A reagent composition having the following composition, which was used as the first reagent in the above-mentioned method for quantifying LDL cholesterol, was prepared.

[0045] Reagent composition PIPES buffer, pH 7.0 50 mmol / L Cholesterol esterase 0.6U / mL Cholesterol oxidase 0.5U / mL Catalase 600U / mL Magnesium chloride 10 mmol / L Surfactant Emulgen B-66 (Kao) 0.2% Bovine serum albumin 1.0%   Hydrogen donor * 1 * 2 (* 1, * 2: In Example 1, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3- Methylaniline (TOOS) was added at 2.00 mM, and in Comparative Example 1, N- (2-hydroxy-3-sulfopro Pill) -3,5-dimethoxyaniline (HDAOS) 0.56 mM)

After measuring the absorbance at 700 nm of each composition, each composition was stored at 8 ° C. for 1 year to obtain 400 to 8
The absorption spectrum at 00 nm and the absorbance at 700 nm were measured. The results are shown in Figure 1 and Table 1.

As shown in FIG. 1, in the reagent composition of Comparative Example 1, the absorption peak showing the color development (peak having maximum absorption at 725 nm) was greatly increased, whereas in the reagent composition of Example 1 Then, it can be seen that the absorption peak is stable and no increase is observed. The HDAOS used as the hydrogen donor in Comparative Example 1 is a compound that has been widely used as a hydrogen donor of a color forming system in the reagent composition of this type. Further, as shown in Table 1, in the reagent composition of Comparative Example 1, the absorbance at the sub-wavelength 700 nm used in the automatic analyzer is significantly increased, whereas in the reagent composition of Example 1, the wavelength is increased. No increase in 700 nm absorbance was observed. From these, it was revealed that with the reagent composition of the present invention, spontaneous color development during storage is significantly suppressed as compared with conventional reagent compositions.

[0048]

[Table 1] Table 1 Influence on measurement wavelength absorbance (700 nm) after storage of reagent composition

Example 2 and Comparative Example 2 A reagent composition having the following composition, which was used as a quantitative reagent for LDL cholesterol, was prepared.

First reagent PIPES buffer, pH 7.0 50 mmol / L cholesterol esterase 0.6 U / mL cholesterol oxidase 0.5 U / mL catalase 600 U / mL magnesium chloride 10 mmol / L surfactant Emulgen B-66 0.2% Bovine serum albumin 1.0% Hydrogen donor * 1 * 2 (* 1, * 2: In Example 2, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) was used. 2.00 mM, in Comparative Example 2 N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS) 0.56 mM)

Second reagent 4-aminoantipyrine 4.0 mmol / L peroxidase 4.0 unit / mL sodium azide 0.05% nonionic surfactant 1.0% (polyoxyethylene alkylphenyl ether)

300 μL of the first reagent preheated to 37 ° C. was mixed with 4 μL of the serum sample and reacted at 37 ° C. for 5 minutes, then 100 μL of the second reagent was added and reacted for 5 minutes, and the absorbance at 600 nm was measured. . The LDL cholesterol was calculated by comparing the measured absorbance with the previously measured absorbance of a known concentration sample.

After storing each composition at 8 ° C. for 1 year, LDL cholesterol in the serum sample was calculated. As a control method, LDL cholesterol in the sample was determined by ultracentrifugation. As the ultracentrifugation method, the method described in "Shinsei Chemistry Laboratory Course Vol. 4, Lipid I Neutral Lipids and Lipoproteins", 181 (1993) was used.

The results are shown in Table 2. As shown in the table, in Comparative Example 2, there are several cases where there is a difference of 5% or more from the ultracentrifugal method. On the other hand, in Example 2, such a deviation example is not seen, and it can be seen that the LDL cholesterol measuring reagent using the reagent composition of the present invention accurately quantifies LDL cholesterol.

[0055]

[Table 2] Table 2 Effect on LDL cholesterol measurement value after storage of reagent product

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, by using the above-mentioned aniline derivative as a hydrogen donor, spontaneous color development during storage can be suppressed, the influence on the change in absorbance by an automatic analyzer can be avoided, and long-term accuracy can be obtained. Various measurements are possible.

[Brief description of drawings]

FIG. 1 Composition of Example 1 (TOOS) and Composition of Comparative Example 1
(HDAOS) shows the absorption spectrum after 1 year storage at 8 ° C.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/92 G01N 33/92 A (72) Inventor Yuko Hirao 1359- 1 Denka Seiken Co., Ltd. Kagamida Plant (72) Inventor Hiroshi Matsui 1359-1 Kagami Kagami, Gokoshi City, Niigata Prefecture Denka Seiken Co., Ltd. Kagamida Plant F-term (reference) 2G045 AA25 BA01 BA11 BB29 BB52 BB54 CA26 DA62 DA64 DA69 DA77 FB01 FB11 GC10 2G054 AA07 AB02 AB05 BA01 BA02 BA03 BA04 BB13 BB20 CA21 CE02 CE08 EA01 EA04 EB01 EB12 GB01 JA09 4B063 QA19 QQ03 QQ22 QQ23 QQ32 QR48 QR66 QS02 QS28 QX02

Claims (3)

[Claims] 1. A reagent composition for forming a dye by the reaction of hydrogen peroxide, a hydrogen donor and a hydrogen acceptor, and quantifying the formed dye, comprising at least the hydrogen donor. A reagent composition containing albumin at the same time, wherein the hydrogen donor is an aniline derivative in which an alkoxyl group is not bonded to both the 3-position and the 5-position. 2. The composition according to claim 1, which is used in a method for quantifying cholesterol in low density lipoprotein. 3. The aniline derivative has the following general formula (I):
The composition according to claim 1 or 2, which has a structure represented by: [Chemical 1] (However, R ¹ and R ² each independently represent a hydrogen atom or an alkyl group, R ³ represents a sulfoalkyl group or a hydroxysulfoalkyl group, and R ⁴ represents a hydrogen atom or an alkyl group.)