JPH0731498A - Determination kit for 1,5-anhydroglucitol and determination method using the kit - Google Patents

Abstract

PURPOSE:To provide the subject kit which comprises a specific pretreating reagent, a coloring reagent and a solvent, and contains a color-developing agent for detection and reagents for color-development, uses body fluid as a sample and is useful in diagnosis of diabetes because it can do measurement of high accuracy in a low concentration region with high sensitivity and has excellent storability. CONSTITUTION:(A) This kit comprises a freeze-dried pre-treatment reagent, a freeze-dried coloring reagent and a solvent dissolving both reagents; (B) the color-developing agent for detection comprises 2-hydrazono-2,3-dihydro-3-methyl-6- benzothiazolesulfonic acid, an aniline derivative and peroxidase; (C) the enzyme to be used for coloring reagent is pyranose oxidase; (D) the enzyme for the pretreatment reagent is an enzyme converting the saccharides in the sample into structure which do not react with the pyranose oxidase and (E) one or more components of the color-developing agent are combined with the pre- treatment reagent or the coloring reagent and the kit uses body fluid as a sample to be measured.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a kit for quantifying 1,5-anhydroglucitol and a quantification method using the kit, which is used for clinical diagnosis of diabetes.

[0002]

2. Description of the Related Art 1,5-Anhydroglucitol is a clinically important test in recent years as a useful diagnostic marker for diabetes because of its low concentration in body fluids such as serum, plasma and urine associated with diabetes. Therefore, it is strongly desired to develop a quantitative kit applicable to a commercially available automatic analyzer.

Conventionally, quantification of 1,5-anhydroglucitol has been carried out by gas chromatography, but recently, pyranose oxidase (EC1.1.3.10), L-sorbose oxidase (EC1.1.3.11), etc. Has been developed, and a method for colorimetric determination has been developed using hydrogen peroxide generated in this case. When using the above-mentioned enzymes and quantifying 1,5-anhydroglucitol using a body fluid, for example, serum as a sample, this kind of enzyme has low substrate specificity and is a sugar present in the sample. In order to react mainly with glucose, it is necessary to perform a pretreatment to prevent the interfering reaction. As a method for preventing the interference by sugars, a method of physically removing using a column (Japanese Patent Laid-Open No. 63-18
5397) and a method of converting a saccharide into a structure that does not react with pyranose oxidase, L-sorbose oxidase and the like. This latter saccharide conversion treatment generally utilizes an enzymatic reaction, and glucose oxidase (JP-A-1-320998), hexokinase (JP-A-2-104298), or glucokinase (JP-A-2-104998) is used as a converting enzyme. 3-27299 gazette) is proposed.

[0004]

[Problems to be Solved by the Invention or Objects of the Invention] A kit for quantifying 1,5-anhydroglucitol, which utilizes the former method for removing sugars, is available from Nippon Kayaku Co., Ltd.
It is already on the market with the product name "G". The method using this kit is to remove sugars, mainly glucose, to a disposable (disposable) column.
(Serum or plasma) and then water several times.
It is carried out by eluting 1,5-anhydroglucitol, adding a coloring reagent to the eluate, reacting for 1 hour, and then performing colorimetric determination. This method is complicated to operate and requires about 1 hour and 30 minutes for all operations, so it is difficult to apply it to an automatic analyzer, and there is a problem in routine measurement work. On the other hand, a method using a pretreatment method for converting the structure of saccharides using an enzyme is expected to be applied to an automatic analyzer, but has not been put to practical use in practice. It is presumed that this is due to the detection sensitivity in colorimetric determination. That is, the blood glucose concentration is about 100 mg / dl in a healthy person and reaches 1000 mg / dl or more in a diabetic patient, but the concentration of 1,5-anhydroglucitol is in a healthy person. It is about 14 μg / ml or more (about 1/100 of glucose), and there is a special circumstance that diabetic patients lower it below 10 μg / ml unlike glucose. Further, in order to apply it to an automatic analyzer, it is necessary to reduce the amount of the sample as much as possible because there is a limit to the ability of the enzyme to convert the structure of a large amount of glucose within a certain time by the enzyme treatment. However, in such cases, the sensitivity of measurement in the low concentration range tends to decrease, and the conventional 1,5-anhydroglucan including a pretreatment step that blocks the interfering reaction by using the enzyme to convert the saccharide structure is used. The citrate assay is applicable to instrumental analysis in the high concentration range (e.g. 30-50 μg / ml or higher), but at the low concentration range (e.g. 0-10 μg) required in the field of clinical testing.
This is probably because it could not be applied to high-precision measurement at (/ ml).

Therefore, the basic object of the present invention is to provide a quantitative method capable of highly accurately measuring 1,5-anhydroglucitol by an automatic analyzer even in the low concentration range required in clinical tests. It is to establish, and a more specific object is to provide a quantification kit for carrying out the quantification method.

[0006]

According to the present invention, the above-mentioned specific object is to use body fluid as a sample.
-A kit for quantifying anhydroglucitol, which comprises a) a pretreatment reagent that is a freeze-dried product, a color reagent that is a freeze-dried product, and a solution for dissolving each of these reagents. , B) The coloring agent for detection is 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazolesulfonic acid, an aniline derivative and peroxidase, and c) the enzyme for coloring reagent is pyranose. An oxidase, d) an enzyme for the pretreatment reagent that converts a saccharide in the sample into a structure that does not react with the pyranose oxidase, and e) one or more of the above-mentioned color formers. This is accomplished by a kit for quantifying 1,5-anhydroglucitol in which the components are combined with a pretreatment reagent or a color reagent.

On the other hand, the above-mentioned basic purpose is a method for quantifying 1,5-anhydroglucitol using the above-mentioned kit, in which the pretreatment reagent is dissolved in the solution for dissolution and added to the body fluid. React for 3-10 minutes, then add a solution of the coloring reagent dissolved in the solution for further reaction for 3-10 minutes, and measure the change in absorbance per unit time during that period.
This is accomplished by a quantitative method of 1,5-anhydroglucitol, which matches the measured value with standard calibration data.

The quantification kit according to the present invention has both the pretreatment reagent and the coloring reagent as freeze-dried products, and the liquid agent is only a solution (buffer solution) for dissolving these reagents, and is therefore simple. It has excellent storability. On the other hand, the quantification method according to the present invention is easy to operate and can be applied to an automatic analyzer because the required time is short, and the measurement sensitivity is high.
It can be measured with high accuracy even when the concentration of 5-anhydroglucitol is low. Therefore, all the above-mentioned problems are completely solved.

Various compounds can be used as the aniline derivative used as one component of the color former, but from the viewpoint of sensitivity, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N- Ethyl-N-
(3-sulfopropyl) aniline (ALPS), N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) aniline (ALOS) or N
-Ethyl-N- (3-sulfopropyl) -3-aniline (TOPS) is preferred. The three components forming the color former can be combined with the pretreatment reagent or the color reagent, but
It is preferred to combine one component with the pretreatment reagent and another component with the color reagent. One preferred example of this is the combination of incorporating 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazolesulfonic acid with a pretreatment reagent and an aniline derivative and peroxidase with a color reagent. 5 to 50 peroxidase in the reaction mixture
The presence of U / ml is sufficient. On the other hand, the concentration of aniline derivative in the reaction solution was 0.2-10 mM and 2-hydrazono-2,3-
The dihydro-3-methyl-6-benzothiazole sulfonic acid concentration is preferably set to 0.1-5 mM. This is because if the concentration is set higher than necessary, the reagent blank becomes large, while if the concentration is too low, the sensitivity becomes insufficient.

As described above, the enzyme constituting the main component of the pretreatment reagent is an enzyme that converts the structure of the saccharide contained in the sample to block the interfering reaction. For example, hexokinase and glucokinase are Can be used. Glucose oxidase and glucose dehydrogenase also exist as enzymes having the above-mentioned actions, and although these enzymes cannot be used, D-glucono-δ-lactone produced by the action of these enzymes on glucose is presented. Pyranose oxidase, which is an enzyme for color reagents, reacts slightly, but is not preferred. The pretreatment reagent may contain a coenzyme and a reaction accelerator known per se necessary for the enzymatic reaction.

The lysing solution used for the preparation of the pretreatment reagent and the coloring reagent, which are lyophilized products, and the buffer, which is a solution for dissolving these reagents at the time of use, have an optimum pH for the enzymatic reaction. There is no particular limitation as long as it can be adjusted to about 5-9, preferably 6.5-8.0.
A hydrochloric acid buffer solution, a phosphate buffer solution or the like can be used.

Incidentally, the additional points relating to the present invention are as follows. When applying the method for quantifying 1,5-anhydroglucitol using pyranose oxidase as an enzyme for coloring reagent to a generally commercially available automatic analyzer, hydrogen peroxide produced by the action of this enzyme is 2-Hydrazono-2,3-as a component of color former for colorimetric determination
Only by using a combination of dihydro-3-methyl-6-benzothiazole sulfonic acid and an aniline derivative, highly sensitive and highly accurate measurement becomes possible, and it becomes a practical one that can be used for clinical laboratory purposes. The color former used in the present invention has been conventionally used for the colorimetric determination of hydrogen peroxide, and it is about twice as much as the combination of 4-aminoantipyrine and aniline derivative when measured under the same conditions. It is considered to be a combination of highly sensitive reagents. This is because, for example, when the pH is 7 and TOOS is selected as the aniline derivative, the molar extinction coefficient of 4-aminoantipyrine in combination with TOOS is about 30,000, and 2-hydrazono-2,3- This is because the molar extinction coefficient in the case of combining dihydro-3-methyl-6-benzothiazolesulfonic acid and TOOS is about 50,000. However, when these color formers were applied to the determination of 1,5-anhydroglucitol, the combination of 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazolesulfonic acid and aniline derivative And the fact that 4-5-antiantipyrine and aniline derivative combinations resulted in a 4-5 fold increase in sensitivity over the combination. The degree of this increase in sensitivity is completely unexpected, and this remarkable increase in sensitivity leads to a reduction in the amount of sample, a decrease in the amount of saccharides that interfere with measurement, and a reduction in the time required for its structural conversion (for example, when hexokinase was used. In some cases, 2000 mg / dl glucose can be structurally converted with a reaction time of 5 minutes), which improves the measurement accuracy in the low concentration range. -It has become possible to quantify anhydroglucitol. In the method of the present invention, the quantification of 1,5-anhydroglucitol is carried out because hydrogen peroxide produced by the action of pyranose oxidase causes the color former to develop a color.
It is carried out by measuring the absorbance of the reaction solution.This absorbance measurement may be performed by using the endpoint method, which measures at the end point of the reaction, or the rate method, which measures the amount of change in absorbance per unit time, for example, 1 minute. Any of these can be used. However, if the end point method is performed after performing the pyranose oxidase reaction for 5 minutes, the pyranose oxidase is added in the reaction solution at 35 U /
ml or more is required, while when using the rate method,
The rate method is adopted in the present invention because a sufficient change in absorbance can be obtained at 2-20 U / ml and the endpoint method has the following problems. When the endpoint method is used, in which the reaction is completed and then measured, the required amount of the enzyme is large because the activity of pyranose oxidase for 1,5-anhydroglucitol is low, and the reaction time Also becomes longer. Since various saccharides are contained in the sample, it is extremely difficult to convert the structure of saccharides that are present in very small amounts, so increasing the amount of enzyme or setting the reaction time longer is a substrate for the enzyme. Since the specificity is not high, an error may occur in the measured value. Therefore, in the present invention, the rate method is adopted, and the effect of trace sugars is minimized by using a small amount of enzyme and performing quantification in a short time. Although the colorimetric quantification by the rate method has the above-mentioned advantages, the measurement sensitivity tends to be lower than that of the endpoint method, but the sensitivity of the color former is extremely high as described above. Is sufficiently supplemented to enable highly accurate quantification.

[0013]

EXAMPLES The present invention will be described in more detail and specifically with reference to the measurement sensitivity comparison test, production examples and test examples. Measurement Sensitivity Comparison Test Example As a color-forming compound, the present invention: 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazolesulfonic acid (measurement wavelength: 570 nm), conventional technology: 4-aminoantipyrine (measurement wavelength : 550 nm) and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) as a color compound are adopted as a common substance. , There are two types of color-forming compounds.
Types) and B were prepared. Reagent A: Compound for color development 1 mM Peroxidase 20 U / ml Tris-HCl buffer (pH 7.5) 0.1 M Reagent B: Pyranose oxidase 80 U / ml TOOS 18 mM Operation and result: 1,5-anhydroglucitol 0, 20
Or, use an aqueous solution containing 50 μg / ml as a sample, and
175 μl of Test Solution A was added to 5 μl and pre-heated at 37 ° C for 5 minutes. Next, add 25 μl of Test Solution B and add 5
The reaction is performed for a minute, and the change in absorbance per minute during that time
(ABS / min) was measured. The results were as shown in Figure 1. The theoretical sensitivity derived from the molar extinction coefficient should be about twice as high as the combination of color formers used in the method of the present invention compared to the case of the prior art method.
The result shown in 1 is about 4.4 times higher.

Production Example A pretreatment reagent, a coloring reagent and a solution for dissolving these reagents were prepared in the following manner, and these were set in a set to produce a kit for quantitatively determining 1,5-anhydroglucitol. . This kit is for 100 measurements. a) Pretreatment reagent 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazole sulfonate 6.0 mg, peroxidase 400 U, hexokinase 400 U, ATP 8.4 mg, phosphoenolpyruvate trisodium 400 mg, pyruvin Acid kinase 470 U
And sucrose 40 mg in 20 mM Tris-HCl buffer (pH 7.5) 2 m
Prepared by adding the resulting solution to the l and dissolving it into a brown bottle and freeze-drying. b) Color reagent N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline
(MAPS) 16.2 mg, pyranose oxidase 240 U and sucrose 20 mg were added to 20 ml Tris-hydrochloric acid buffer solution (pH 7.5) 1 ml, and the dissolved solution was placed in a brown bottle and freeze-dried. c) Pretreatment reagent solution 0.1M Tris-hydrochloric acid buffer solution (pH 7.5) containing 14.4 mM magnesium chloride was prepared and put in a container (20 ml). d) Prepare 0.1M Tris-HCl buffer (pH 7.5) for dissolving color reagent and put it in a container.
4ml was put.

In the body fluid using the above-mentioned assay kit
The measurement operation of 1,5-anhydroglucitol is as follows. In the case of a commercially available automatic analyzer (“COBAS MIRA” commercially available from Nippon Roche Co., Ltd.), add 175 μl of pretreatment reagent to 5 μl of body fluid as a sample, such as serum.
Incubate at 37 ℃ for 5 minutes, then add 25 μl of color reagent and incubate for 5 minutes. During that time, measure the change in absorbance (ABS / min) per minute. 1,5-in sample serum
The anhydroglucitol concentration is a standard calibration curve prepared in advance by measuring the amount of change in absorbance using a 1,5-anhydroglucitol standard solution of known concentration as a sample.
(The type of aniline derivative is different from that of the kit for quantification according to this production example, but Figure 1 of the accompanying drawings showing the results in the above-mentioned “Comparison test for measurement sensitivity” is also a kind of standard calibration curve) Obtain (Of course, when performing measurement using an automatic analyzer, the data corresponding to the standard calibration curve is stored in the instrument and automatically compared with the measured value for the sample).

Test Example 1 (Effect of Glucose) A sample serum was prepared by adding an aqueous glucose solution of various concentrations at a ratio of 1 to 9 kinds of serum 9 and prepared in the latter stage of the above “Production Example”. The influence of glucose on the 1,5-anhydroglucitol concentration value in each sample serum was examined by measuring the change in absorbance according to the procedure described above. The results are shown in Table 1 below,
It was found that even if glucose was present up to 2000 mg / dl in the sample, the structure was converted by the pretreatment and there was almost no effect on the measured value.

[0017]

[Table 1]

Test Example 2 (simultaneous reproducibility) Four kinds of sera having different 1,5-anhydroglucitol concentrations were used as samples, and the change in absorbance was measured according to the operation procedure described in the latter part of the above-mentioned "Production Example". Simultaneous reproducibility of 1,5-anhydroglucitol concentration in each sample serum was obtained by measuring 5 times. The results are shown in Table 2 below, and it was found that the method of the present invention had good co-reproducibility.

[0019]

[Table 2] In Table 2, the units for the measured and average values are μ
It is g / ml.

Test Example 3 (Correlation with other quantitative methods) For 30 kinds of serum samples, a quantitative kit according to the above-mentioned production example and a quantitative kit "Rana AG" commercially available from Nippon Kayaku Co., Ltd. The 1,5-anhydroglucitol concentration in the sample was measured using and (in the case of using the kit according to the present invention, instrumental analysis, and in the case of using a commercially available kit, it was measured by a manual method). The comparison of the measurement results is shown in Fig. 2, and the correlation coefficient was 0.994, indicating that both methods have high correlation.

[0021]

INDUSTRIAL APPLICABILITY The present invention has excellent storage stability,
-Providing a kit for quantifying anhydroglucitol and a quantification method using the kit. In order to measure the substance for the purpose of clinical diagnosis of diabetes, measurement in a low concentration range of 10 μg / ml or less is required, and in the past, this has made instrument analysis extremely difficult or impossible. To meet the clinical requirements.

[Brief description of drawings]

FIG. 1 is a graph showing a comparison of measurement sensitivity between the method of the present invention and the conventional method.

FIG. 2 is a graph showing the correlation between the measurement results obtained by measuring 1,5-anhydroglucitol in serum samples using the quantification kit of the present invention and a commercially available quantification kit.

Claims (6)

[Claims] 1. A kit for quantifying 1,5-anhydroglucitol, which uses a body fluid as a sample, comprising: a) a pretreatment reagent which is a freeze-dried product, and a color reagent which is a freeze-dried product. It is composed of the respective solutions for dissolution of both reagents, and b) the coloring agent for detection is 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazole sulfonic acid, an aniline derivative, and peroxidase. And c) the enzyme for the color reagent is pyranose oxidase, d) the enzyme for the pretreatment reagent is an enzyme that converts the saccharides in the sample into a structure that does not react with the pyranose oxidase, and e) A kit for quantifying 1,5-anhydroglucitol, characterized in that one or more components of the above color former are combined with a pretreatment reagent or a color reagent. 2. The aniline derivative is N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) aniline and N-ethyl-N
The kit for quantifying 1,5-anhydroglucitol according to claim 1, which is selected from-(3-sulfopropyl) -3-aniline. 3. The method according to claim 1 or 2, wherein each of the lysing solutions for the pretreatment reagent and the coloring reagent is a buffer solution and has a pH of 5-9. A kit for quantifying 1,5-anhydroglucitol. 4. A kit for quantifying 1,5-anhydroglucitol in a body fluid, comprising: a) a pretreatment reagent which is a lyophilized product; and a color reagent which is a lyophilized product. It is composed of the respective solutions for dissolution of both reagents, and b) the coloring agent for detection is 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazole sulfonic acid, an aniline derivative, and peroxidase. And c) the enzyme for the color reagent is pyranose oxidase, d) the enzyme for the pretreatment reagent is an enzyme that converts the saccharides in the sample into a structure that does not react with the pyranose oxidase, and e) Use a kit in which one or more of the above color formers are combined with pretreatment or color reagents
In the method for quantifying 1,5-anhydroglucitol, the pretreatment reagent was dissolved in the lysis solution and added to the body fluid.
-React for 10 minutes, then add a solution of color reagent dissolved in the solution for reaction, and react for another 3-10 minutes,
The amount of change in absorbance per unit time during that period is measured, and the measured value is collated with standard calibration data, 1,5-
Determination of anhydroglucitol. 5. The method for quantifying 1,5-anhydroglucitol according to claim 4, wherein the pyranose oxidase concentration in the reaction solution is set to 2-20 U / ml. 6. The aniline derivative is N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) aniline and N-ethyl-N
It was selected from-(3-sulfopropyl) -3-aniline, and the concentration of aniline derivative in the reaction solution was 0.2-10 mM.
And the concentration of 2-hydrazono-2,3-dihydro-3-methyl-6-benzothiazole sulfonic acid is set to 0.1-5 mM, 1,5-An according to claim 4 or 5. Determination of hydroglucitol.