JPH0145360B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a stable glycoside composition, and more particularly to a glycoside useful as a substrate for a reagent for measuring α-amylase activity using a coupled enzyme method. (Prior art) Measurement of α-amylase activity in body fluids such as serum, urine, and pancreatic juice has important significance in clinical diagnosis, and is particularly useful for acute or chronic pancreatitis, pancreatic cancer, and even It is an essential measurement item in the differential diagnosis of mumps, etc. Among the conventionally proposed methods for measuring α-amylase activity, the coupled enzyme method has recently attracted attention. As the coupled enzyme method, in particular, JP-A-53-11092,
Light-absorbing and light-sensitizing agents typically containing p-nitrophenol and 4-methylumbelliferone at the reducing end of oligosaccharides as disclosed in JP-A-54-25893 and JP-A-54-51892 A method using a glycoside in which a compound having a phenolic hydroxyl group is glycosidic bonded as a substrate is particularly excellent and has been put into practical use. Also, compounds having a phenolic hydroxyl group such as 2,4-dichlorophenol at the reducing end of an oligosaccharide as disclosed in JP-A-56-35998, for example, oxidized with 4-aminoantipyrine etc. A method of using oligosaccharide aglycones with glycoside bonds that produce colored pigments by condensation has also been put into practical use. (Problems to be Solved by the Invention) However, α-
In the amylase activity measurement method, the solution stability of the substrate is not sufficient, and when used for several days or weeks, the substrate decomposes during storage, resulting in an increase in reagent blanks and furthermore, an insufficient amount of substrate. Sometimes. (Means for Solving the Problem) The present inventors conducted various studies for the purpose of stabilizing a solution containing a substrate, and found that the substrate was significantly stabilized when a specific concentration of nonionic surfactant was added. , arrived at the present invention. That is, the present invention uses 0.02 to 100 mg/ml of a glycoside in which an aromatic compound having a hydroxyl group is bound to the reducing terminal hydroxyl group of a malto-oligosaccharide having 1 to 10 repeating units to the entire composition and a nonionic surfactant. α- characterized in that it consists of a buffer solution or water containing 0.2% by volume or more based on the total composition.
This is a substrate solution for measuring amylase. The glycoside in the present invention has a repeating unit number of 1 to
An aromatic compound having a hydroxyl group is glycosidic bonded to the reducing terminal hydroxyl group of the maltooligosaccharide (10). The glycosidic bond is α-
It may be either a bond or a β-bond. Examples of malto-oligosaccharides include maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose, and particularly maltotetraose, maltopentaose, maltohexaose, and maltoheptaose. preferable. In the present invention, the aglycone bonded to the malto-oligosaccharide may be any aglycone as long as it is liberated by the action of glucosidase, develops a color when liberated, or is easily quantified, but in particular, it may be an aromatic compound having a hydroxyl group. be. Examples of aromatic compounds having a hydroxyl group bonded to the reducing end of the maltooligosaccharide of the present invention via an α- or β-bond include halogen atoms, hydroxyl groups, alkyl groups having 1 to 6 carbon atoms, alkoxy phenols having a group, an alkoxycarbonyl group or a nitro group, such as chlorophenol, dichlorophenol, hydroxyphenol, alkylphenol, alkoxyphenol, benzoic acid or nitrophenol, halogenated nitrophenol, alkylated nitrophenol, alkoxylated nitrophenol. Examples include phenol, nitrated benzoic acid, dinitrophenol, and the like. It may also be an aromatic compound such as 4-methyl-umbelliferone. In particular phenols having at least one nitro group, such as 4-nitrophenol, 2-chloro-4-nitrophenol, 2,6-dichloro-
4-nitrophenol, 2,6-dibromo-4-
Nitrophenol, 2-bromo-4-nitrophenol, 2-nitrophenol, 2-hydroxy-4-nitrophenol, 3-hydroxy-4-
Nitrophenol and the like are preferred. Next, the glycosides of the present invention will be further explained using specific examples. Examples include the following. α-Phenylpentaoside, α-2-chlorophenylpentaoside, α-2,6-dichlorophenylpentaoside, α-4-nitrophenylpentaoside, α-2-chloro-4- Nitrophenylpentaoside, α-2-methyl-4-
Examples include nitrophenyl pentaoside, and β-conjugates thereof, as well as tetraoside, hexaoside, and pentaoside. It may be a mixture of these. Malto-oligosaccharides have substituents such as halogens, alkoxycarbonyl groups, sulfonyl groups bonded with aliphatic or aromatic groups, carbonyl groups bonded with aliphatic or aromatic groups, phenyl groups or glucose residues bonded with 1,6- Malto-oligosaccharides may also be used,
It also includes partially modified maltooligosaccharide chains, such as those in which two hydroxyl groups are intramolecularly cross-linked. In the present invention, glycosides are
The content is 0.2 to 100 mg/ml, preferably 0.5 to 10 mg/ml. The Michaelis constant (Km) value of α-amylase with respect to the substrate is approximately 0.3mM, and is usually 3 to 10 Km.
The standard is to measure at twice the substrate concentration, but the concentration in the substrate solution used for test analysis is preferably 1 to 20 times this. The nonionic surfactant used in the present invention may be any surfactant as long as it has an HLB value in the range of 11 to 18, such as polyoxyethylene-p-isooctylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene Examples include lauryl ester, polyoxyethylene-p-nonylphenyl ether, and polyoxyethylene cetyl ether. In the present invention, the nonionic surfactant is contained at 0.2% by volume or more, preferably from 0.3 to 1.2% by volume, based on the total composition. Usually, in clinical test reagents for measuring body fluids such as blood, in order to reduce the influence of turbidity caused by lipids, proteins, etc. in the body fluids,
Although it is common practice to add a surfactant, the amount of surfactant added for this purpose is usually less than 0.2% by volume. In the present invention, the glycoside that is the substrate can be stabilized by containing the nonionic surfactant in an amount of 0.2% by volume or more based on the total composition. Stabilization cannot be achieved at less than 0.2% by volume. The composition of the present invention contains a buffer or water in addition to the glycoside and nonionic surfactant. In the present invention, a nonionic surfactant may be added to a buffer solution or water in which a glycoside is dissolved, or a glycoside may be added to a buffer solution or water in which a nonionic surfactant is dissolved. Furthermore, the glycoside and the nonionic surfactant may be added to the buffer or water at the same time. The glycoside composition of the present invention may optionally include a coloring agent such as 4-aminoantipyrine, an enzyme,
For example, it may contain α-glucosidase, β-glucosidase, etc., and an oxidizing agent such as sodium periodate. The glycoside composition of the present invention can be used as a substrate for measuring α-amylase activity in body fluids. (Effects of the Invention) In the present invention, the stability of glycosides can be improved by containing the nonionic surfactant in an amount of 0.2% by volume or more based on the total composition. Especially when the nonionic surfactant is 0.1% by volume, the number of storage days (25℃)
While stability is lost after 7 days, when the nonionic surfactant is 0.2% by volume or more, it can be stored stably for 7 days or more. (Example) The present invention will be described below with reference to Examples. Example 1 β-2,4-
Dichlorophenyl maltopentaoside
4 μmole/ml and 4-aminoantipyrine
A substrate-containing buffer was obtained by adding 4 μmole/ml. Next, 0.6% by volume or 1.2% by volume of Triton-X (polyoxyethylene isooctyl phenyl ether) or a nonionic surfactant shown in Table 1 was added to prepare a substrate reagent solution. In order to measure the stability of the obtained substrate reagent solution, absorbance was measured by the following procedure using the following enzyme reagent solution and coloring solution. Reagent composition Enzyme reagent Pipes buffer 0.05M (PH6.7) α-glucosidase 100u/ml β-glucosidase 10u/ml Triton X-100 (polyoxyethylene isooctyl phenyl ether) 0.1% Color developer borate buffer 0.1M (PH8.4) 10 mmol of sodium periodate aqueous solution/20 μ of the sample was placed in a test tube, mixed with 0.5 ml of enzyme test solution, and heated at 37° C. for about 5 minutes. Then the substrate reagent
After adding 0.5ml and warming at 37℃ for exactly 10 minutes,
After adding 2.0 ml of coloring solution and leaving it for 5 minutes, the absorbance was measured at a wavelength of 500 nm. This procedure was repeated when using 20μ of water instead of the sample (reagent blank) and when using an aqueous solution containing 2 mmol/2,4-dichlorophenol (standard), and from the obtained absorbance, the following formula was used. Therefore, the α-amylase value was determined by calculation. Amylase activity value (u/) = Absorbance of sample - Absorbance of reagent blank / Absorbance of standard - Absorbance of reagent blank x 2000 x 1/10 Changes in the reagent blank when the various substrate reagents mentioned above were stored at 4℃ and 25℃. Shown in Table 1. The increase in reagent blank over time indicates that the substrate (β-
This corresponds to the fact that 2,4-dichlorophenyl maltopentaoside) is decomposed and 2,4-dichlorophenol is produced as a result. Therefore, it is clear that the glycosides in the test solution according to the present invention are stabilized.

【table】

【table】

【table】

Claims (1)

[Claims] 1 A glycoside in which an aromatic compound having a hydroxyl group is bound to the reducing terminal hydroxyl group of a maltooligosaccharide having 1 to 10 repeating units is added to the entire composition.
1. A substrate solution for α-amylase measurement, comprising a buffer solution or water containing 0.2 to 100 mg/ml and a nonionic surfactant in an amount of 0.2% by volume or more based on the total composition.