JP2002355097A - Reagent for measuring alpha-amylase activity and measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for measuring α-amylase activity without affected by the influence of the hypoglycemic agent in a sample and provide a method for measuring α-amylase activity. SOLUTION: Alfa-amylase activity in a biological sample is measured by measuring a measurable material isolated from a malto-oligosaccharide derivative by reacting an agent containing a malto-oligosaccharide derivative expressed by general formula (wherein R<1> expresses a group which binds to 1 position of a reduced terminal glucose through a bond capable of being cleaved with α-amylase and becomes a material to be detected when the bond is cleaved) and α-glucosidase with a biological sample containing α-amylase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing α-
The present invention relates to a reagent for measuring amylase activity and a method for measuring α-amylase activity.

[0002]

2. Description of the Related Art Amylase is a digestive enzyme mainly secreted from the pancreas and salivary glands. The activity of α-amylase in blood and urine is measured by measuring pancreatic diseases such as acute pancreatitis and salivary glands such as epidemic parotitis. It is used to diagnose diseases. As a method for measuring the activity of α-amylase, a group that becomes a measurable substance after being cleaved by α-amylase at the 1-position of glucose at the reducing end is used as an aglycone, and β- or 4-position of glucose at the non-reducing end is β-amylase. Using a maltooligosaccharide derivative having a galactopyranosyl group and having 2 to 4 glucose units as a substrate, the substrate is reacted with α-amylase in a sample in the absence of α-glucosidase, and the released measurement is performed. A method of quantifying a possible substance is known (Japanese Patent No. 2807949). However, this method is disadvantageous in that when a hypoglycemic agent is present in a biological sample, the α-amylase activity value is reduced as compared with the case where a biological sample not containing a hypoglycemic agent is used due to the effect of the substance. have.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a reagent for measuring α-amylase activity and a method for measuring α-amylase activity which are not affected by a hypoglycemic agent in a biological sample.

[0004]

The present invention provides the following (1) to
Regarding (6). (1) General formula (I)

[0005]

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(Wherein, R ¹ represents a group that binds to the reducing terminal at position 1 via a bond that can be cleaved by α-amylase and becomes a measurable substance when the bond is cleaved). A reagent for measuring α-amylase activity in a biological sample, comprising the maltooligosaccharide derivative and α-glucosidase. (2) The reagent according to (1), wherein the measurable substance is a substance which itself exhibits absorbance or a fluorescent substance.

(3) The reagent according to (1) or (2), wherein R ¹ is a 2-chloro-4-nitrophenyl group. (4) General formula (I)

[0008]

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(Wherein, R ¹ represents a group which binds to glucose 1 at the reducing end via a bond which can be cleaved by α-amylase and becomes a measurable substance when the bond is cleaved) Reacting a reagent containing the represented maltooligosaccharide derivative and α-glucosidase with a biological sample containing α-amylase, and measuring the amount of a measurable substance released from the maltooligosaccharide derivative. A method for measuring α-amylase activity in a biological sample.

(5) The method according to (4), wherein the measurable substance is a substance which itself exhibits absorbance or a fluorescent substance. (6) The method according to (4) or (5), wherein R ¹ is a 2-chloro-4-nitrophenyl group.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION α-Amylase Substrate The α-amylase substrate used in the present invention is represented by the general formula (I)

[0012]

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(Wherein, R ¹ represents a group which binds to glucose 1 at the reducing end via a bond which can be cleaved by α-amylase and becomes a measurable substance when the bond is cleaved) The maltooligosaccharide derivative [hereinafter referred to as maltooligosaccharide derivative (I)] is used. Maltooligosaccharide derivative (I) has maltose as a basic structure, and β-position is added to glucose 4 at the non-reducing end of maltose.
A galactopyranosyl group is O-glycosidically linked, and has a group R ¹ which is cleaved by α-amylase to be a measurable substance at the reducing end of maltose;
R ¹ is bonded to the glucose 1 position at the reducing end via an α-O-glycoside bond. R ¹ is, for example, 4
A substituted phenyl group such as -nitrophenyl group, 2-nitrophenyl group, 2-chloro-4-nitrophenyl group, and 2,4-dichlorophenyl group; and a fluorescent group such as 4-methylumbelliferyl group. Is preferably a 2-chloro-4-nitrophenyl group.

Examples of the maltooligosaccharide derivative (I) include, for example, 2-chloro-4-nitrophenyl 4-O-α-
D-galactopyranosyl-α-maltoside, 4-nitrophenyl 4-O-α-D-galactopyranosyl-α-
Maltoside and the like;

[0015]

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2-chloro-4-nitrophenyl 4-O-β-D-galactopyranosyl-α-maltoside [hereinafter referred to as maltooligosaccharide derivative (II)] is preferably exemplified. 2. Synthesis of Maltooligosaccharide Derivative (II) Maltooligosaccharide derivative (II) was obtained from a commercially available compound.
It can be synthesized by a general synthesis method. For example, the method described in Japanese Patent No.
The compound can be synthesized by the method described in U.S. Pat.

3. α-Glucosidase The α-glucosidase used in the present invention has the enzyme number [EC 3.2.1.20. And α
Exoglucosidase that hydrolyzes glucosidic bonds. In the present invention, not only α-glucosidase derived from animals, plants or microorganisms, but also α-glucosidase modified by genetic engineering techniques can be used. In the present invention, since α-glucosidase is used, α-amylase activity in a biological sample can be measured without being affected by a hypoglycemic agent present in the biological sample.

4. Biological Sample The biological sample used in the present invention includes, for example, whole blood, plasma, serum, sweat, saliva, urine, pancreatic fluid, amniotic fluid, cerebrospinal fluid and the like, and preferably plasma, serum and urine. Since the measurement method of the present invention is not affected by the hypoglycemic agent, the present invention can also use a biological sample collected from a patient who has been administered a hypoglycemic agent due to diabetes or the like.

5. Hypoglycemic agents Examples of hypoglycemic agents which do not affect the method for measuring α-amylase of the present invention include O-4,6-dideoxy-4.
-{[(1S, 4R, 5S, 6S) -4,5,6-trihydroxy-3- (hydroxymethyl) -2-cyclohexen-1-yl] amino} -α-D-glucopyranosyl- (1 → 4 ) -O-α-D-glucopyranosyl- (1
→ 4) -D-glucopyranose or (+)-1 _L- [1
(OH), 2,4,5 / 3] -5- [2-hydroxy-
1- (hydroxymethyl) ethyl] amino-1-C-
Examples include, but are not limited to, sugar absorption delaying agents such as (hydroxymethyl) -1,2,3,4-cyclohexanetetraol.

6. Reagent for measuring α-amylase activity The reagent for measuring α-amylase activity of the present invention contains the above maltooligosaccharide derivative (I) and α-glucosidase, and optionally contains an aqueous solution, a surfactant, and a stabilizer. , A preservative, a reaction accelerator and the like. Α of the present invention
-The reagent for measuring amylase activity may be in a lyophilized state or in a state previously dissolved in an aqueous medium. When α-amylase activity is measured using the lyophilized reagent, the lyophilized reagent is used after dissolving in an aqueous medium. The concentration of the maltooligosaccharide derivative (I) in the reagent for measuring α-amylase activity dissolved in an aqueous medium is not particularly limited as long as it is a concentration suitable for measuring α-amylase activity, but is preferably 0.1 to 100mmo
1 / L, more preferably 1.0 to 20 mmol /
L. The concentration of α-glucosidase in the reagent for measuring α-amylase activity dissolved in an aqueous medium is not particularly limited as long as it is a concentration suitable for measuring α-amylase activity, but is preferably 0.5 to 100 U / ML, more preferably 1-20 U / mL.

7. α-Amylase activity measurement kit α-amylase activity measurement reagent is stored in the form of a kit,
May be used. The form of the kit is not particularly limited, and examples thereof include a two-reagent system and a three-reagent system, and a two-reagent system is preferable. Examples of the kit for measuring α-amylase activity using a two-reagent system include a kit comprising a reagent containing a maltooligosaccharide derivative (I) and a reagent containing α-glucosidase. Each reagent constituting the kit for measuring α-amylase activity may contain an aqueous medium, a surfactant, a stabilizer, a preservative, a reaction accelerator, and the like, if necessary.

The reagents constituting the kit for measuring α-amylase activity may be in a lyophilized state or in a state previously dissolved in an aqueous medium. When α-amylase activity is measured using the lyophilized reagent, the lyophilized reagent is used after dissolving in an aqueous medium. α
-The maltooligosaccharide derivative (I) in each reagent constituting the kit for measuring amylase activity has a concentration of preferably 0.1 to 100 mmol / mol when dissolved in an aqueous medium.
L, more preferably 1.0 to 20 mmol
/ L. The α-glucosidase in each reagent constituting the kit for measuring α-amylase activity has a concentration of preferably 0.5 to 100 U / mL in a state of being dissolved in an aqueous medium, more preferably 1 to 100 U / mL. It is contained so as to be 20 U / mL.

The aqueous medium is not particularly limited, and includes, for example, deionized water, distilled water, and a buffer, but a buffer is preferred. The buffer used for the buffer is not particularly limited as long as it has a buffering ability and stably dissolves maltooligosaccharide derivative (I) and α-glucosidase.
For example, 2-morpholinoethanesulfonic acid (MES) having a pH of 6.0 to 9.0, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-tr)
is), N- (2-acetamido) iminodiacetic acid (AD
A), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2
-Aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPS
O), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TE
S), N- (2-hydroxyethyl) piperazine-N '
-2-ethanesulfonic acid (HEPES), 3- [N, N
-Bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 2-hydroxy-3- {N- [tris (hydroxymethyl) methyl] methyl
Amino dipropanesulfonic acid (TAPSO), piperazine-N, N'-bis (2-hydroxypropane-3-sulfonic acid) (POPSO), N- (2-hydroxyethyl) piperazine-N'-2-hydroxypropane- 3-
Sulfonic acid (HEPPSO), N- (2-hydroxyethyl) piperazine-N′-3-propanesulfonic acid [(H) EPPS], N-tris (hydroxymethyl)
Methylglycine (Tricine), N, N-bis (2
-Hydroxyethyl) glycine (Bicine), 3-
{N- [tris (hydroxymethyl) methyl] amino}
Good buffering agents such as propanesulfonic acid (TAPS) and the like. The concentration of the buffer is not particularly limited as long as it is a concentration suitable for measurement, but is 0.001 to 2.0 mol / L.
Is preferable, and 0.05 to 1.0 mmol / L is more preferable.

The surfactant includes, for example, a cationic surfactant, an anionic surfactant, an amphoteric surfactant or a nonionic surfactant. As a stabilizer,
For example, ethylenediaminetetraacetic acid (EDTA) and the like can be mentioned. As preservatives, for example, sodium azide,
Antibiotics and the like. Examples of the reaction accelerator include potassium thiocyanate, calcium ion, chloride ion and the like.

8. Method for measuring α-amylase activity The method for measuring α-amylase activity of the present invention includes reacting a reagent containing a maltooligosaccharide derivative (I) and α-glucosidase with a biological sample containing α-amylase to obtain the maltooligosaccharide. There is no particular limitation as long as it is a method for quantifying a measurable substance released from the derivative (I).

The reaction between the reagent containing the maltooligosaccharide derivative (I) and α-glucosidase and the biological sample containing α-amylase can be carried out under the conditions of a usual enzyme reaction. The reaction temperature in the reaction is 10
To 50 ° C, more preferably 20 to 40 ° C,
5-37 ° C is particularly preferred. The reaction time in the reaction is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 5 to 15 minutes. The concentration of the maltooligosaccharide derivative (I) in the reaction is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.1 to 1
00 mmol / L, more preferably 1.0 to 20
mmol / L. The concentration of α-glucosidase in the reaction is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.5 to 100 U / mL, more preferably 1 to 20 U / mL.

The α-amylase activity measurement of the present invention can be performed, for example, by using α-amylase activity measuring reagent as α-amylase activity reagent.
Using a two-reagent kit comprising a reagent containing glucosidase [hereinafter referred to as reagent (1)] and a reagent containing maltooligosaccharide derivative (I) [hereinafter referred to as reagent (2)], Can be done as follows. First, the reagent (1) is added to a biological sample containing α-amylase to cause a reaction, and then the reagent (2) is added to the reaction solution, the amount of released aglycone is determined, and the obtained quantitative value is determined in advance. Based on the prepared calibration curve (calibration curve correlating the concentration of a measurable substance and α-amylase activity), the α-
Amylase activity is measured. In the measurement of α-amylase activity, the reagent (1) and the reagent (2) may be added to the biological sample at the same time, but a method of adding the reagent (2) after adding the reagent (1) to the biological sample Is preferred.

Examples of a method for quantifying the released measurable substance include an absorbance method and a fluorescence method. The quantification of the released measurable substance is, for example, by measuring the absorbance of the substance when the released substance itself shows absorbance, and when the substance is a fluorescent substance,
It can be performed by measuring the fluorescence of the substance. In particular, when a maltooligosaccharide derivative (II) is used as a substrate of α-amylase, 2-chloro-4-nitrophenol is released as a measurable substance. Therefore, quantification of the phenol derivative can be performed, for example, by measuring the absorbance at 405 nm. Can be measured.

As a method for quantifying α-amylase activity, both a rate method obtained by continuously quantifying released aglycone and an endpoint method obtained by quantifying released aglycone after reacting for a certain period of time are used. Can be used. The α-amylase activity measuring reagent and the measuring method of the present invention are not limited to the measurement of α-amylase activity in a sample, and the measurement of calcium ions, chloride ions and the like in a sample incorporating the α-amylase activity measuring method of the present invention. Is also applicable.

Examples of the present invention will be described below, but the present invention is not limited to these examples. The reagents and enzymes used here were from the following manufacturers. MES buffer (manufactured by Dojin Kagaku), calcium acetate monohydrate (manufactured by Kanto Kagaku), sodium chloride (manufactured by Wako Pure Chemical Industries), maltooligosaccharide derivative (II) (2-chloro-4-nitrophenyl 4 —O-β-D-galactopyranosyl-α-maltoside) (manufactured by Toyobo Co., Ltd.), potassium thiocyanate (manufactured by Kanto Kagaku), α-glucosidase (manufactured by Unitika),
O-4,6-dideoxy-4-{[(1S, 4R, 5
S, 6S) -4,5,6-trihydroxy-3- (hydroxymethyl) -2-cyclohexen-1-yl] amino} -α-D-glucopyranosyl- (1 → 4) -O-α
-D-glucopyranosyl- (1 → 4) -D-glucopyranose (Acarbose ^™ ; manufactured by Bayer AG).

[0031]

Example 1 α-glucosidase-containing α
-Reagent for measuring amylase activity As described below, a reagent for measuring α-amylase activity containing α-glucosidase was prepared as a kit. First reagent MES buffer (pH 6.0) 50 mmol / L Calcium acetate monohydrate 6.7 mmol / L Sodium chloride 400 mmol / L α-glucosidase 10 U / mL Second reagent MES buffer (pH 6.0) 50 mmol / L Potassium thiocyanate 560 mmol / La Maltooligosaccharide derivative (II) 10 mmol / L Comparative Example 1 Reagent for measuring α-amylase activity not containing α-glucosidase As described below, a reagent for measuring α-amylase activity not containing α-glucosidase is kit Prepared as First reagent MES buffer (pH 6.0) 50 mmol / L Calcium chloride monohydrate 6.7 mmol / L Sodium chloride 400 mmol / L Second reagent MES buffer (pH 6.0) 50 mmol / L Potassium thiocyanate 560 mmol / L Maltooligosaccharide derivative (II) 11.2 mmol / L

Example 2 Measurement of α-amylase activity Using the kits prepared in Example 1 and Comparative Example 1, α-amylase activity in an Acarbose ^™ -containing sample was measured. Acarbose ^TM to sample saliva amylase (1500IU / L)
To 0, 0.08, 0.16, 0.24, 0.3 respectively
2. A sample was added to be 0.40 mg / mL. Measurement method 2.25 m of first reagent prepared in Example 1 and Comparative Example 1
To L, 0.05 mL of a sample was added and incubated at 37 ° C. for 5 minutes. 0.75 mL of the second reagent prepared in Example 1 and Comparative Example 1 was added to each solution,
For 5 minutes, the change in absorbance at 405 nm was measured, and the change in absorbance per minute was calculated. This change in absorbance was converted to an amylase activity value using the molecular extinction coefficient of 2-chloro-4-nitrophenol in the composition. As a blank, the same measurement was performed using purified water instead of the sample. The results are shown in Table 1.

[0033]

[Table 1]

Thus, it was found that the reagent of the present invention was not affected by α-amylase even in the presence of a hypoglycemic agent.

[0035]

According to the present invention, a reagent and a method for measuring α-amylase activity which are not affected by a hypoglycemic agent in a sample are provided.

Claims (6)

[Claims] 1. A compound of the general formula (I) (Wherein, R ¹ is bonded to glucose 1 at the reducing end via a bond that can be cleaved by α-amylase and represents a group that becomes a measurable substance when the bond is cleaved). A reagent for measuring α-amylase activity in a biological sample, comprising a maltooligosaccharide derivative and α-glucosidase. 2. The reagent according to claim 1, wherein the measurable substance is a substance which itself exhibits absorbance or a fluorescent substance. 3. The reagent according to claim 1, wherein R ¹ is a 2-chloro-4-nitrophenyl group. 4. A compound of the general formula (I) (Wherein, R ¹ is bonded to glucose 1 at the reducing end via a bond that can be cleaved by α-amylase and represents a group that becomes a measurable substance when the bond is cleaved). Reacting a reagent containing a maltooligosaccharide derivative and α-glucosidase with a biological sample containing α-amylase, and measuring the amount of a measurable substance released from the maltooligosaccharide derivative. For measuring α-amylase activity. 5. The method according to claim 4, wherein the measurable substance is a substance which itself exhibits absorbance or a fluorescent substance. 6. The method according to claim 4, wherein R ¹ is a 2-chloro-4-nitrophenyl group.