JPS6150990A - Stable glycoside composition - Google Patents

Abstract

PURPOSE:A stable glycoside composition, containing a glycoside and a nonionic surfactant in a specific proportion respectively in a buffer solution or water, useful as a substrate for measuring reagent for alpha-amylase activity by the conjugated enzymic method. CONSTITUTION:A stable glycoside composition obtained by adding (A) 0.2-100 mg/ml, preferably 0.5-10mg/ml glycoside, preferably beta-2,4-dichlorophenylmaltopentoxide, and (B) >=0.2vol%, preferably 0.3-1.2vol% nonionic surfactant, preferably having 11-18HLB value, e.g. polyoxyethylene isooctyl phenyl ether, and if necessary (C) a color former, e.g. 4-aminoantipyrine, to a buffer solition, e.g. 0.05M PIPES buffer solution of 6.7pH, or water.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field 1) The present invention relates to a stable glycoside composition, and more particularly to a substrate for a reagent for measuring α-amylase activity using a coupled enzyme method. The present invention relates to glycosides useful as glycosides.

(Prior art) The α-amylase activity in body fluids such as serum, urine, and pancreatic juice has important clinical significance, especially in acute or chronic pancreatitis, pancreatic cancer, Furthermore, it has become an essential measurement item in the differential diagnosis of mumps, etc. Among the previously proposed methods for measuring α-amylase activity, the coupled enzyme method has recently attracted attention. There is. As the conjugate enzyme method, in particular, JP-A-53-11092, JP-A-54
Light-absorbing and light-sensitive phenolic compounds, such as p-nitrophenol and 4-methylumbelliferone, are added to the reducing end of the oligocyclic chain as disclosed in Japanese Patent Application Laid-Open No. 54-25893 and Japanese Patent Application Laid-Open No. 54-51892. A method using a glycoside in which a compound having a hydroxyl group is glycosidic bonded as a substrate is particularly excellent and has been put into practical use. Also % Kaian 5
Oligos as disclosed in Publication No. 6-35998,
Compounds that have a phenolic hydroxyl group such as 2,4-dichlorophenol at the reducing end of the tang, for example, can be oxidized and condensed with 4-aminoantipyrine to produce a colored pigment, resulting in glycosidic bonds and oligosaccharide aglycones. A method for using it as a method has also been put into practical use.

(Problems to be Solved by the Invention) In the α-amylase activity measurement method, the solution stability of the substrate is not sufficient, and when used for several days or weeks, the The substrate may decompose during this process, resulting in an increase in reagent crank and even a lack of substrate amount.

The inventors of the present invention conducted various studies for the purpose of stabilizing a solution containing a substrate, and found that the substrate was significantly stabilized when all nonionic surfactants were added at a specific concentration. Heading, reaching the present invention.

That is, the present invention provides 0.2 to 10019 for the entire composition.
The present invention is a stable glycoside composition characterized in that it consists of a buffer solution or water containing 0.2 volumes or more of a nonionic surfactant based on the total composition.

The atom in the present invention is one in which a compound other than glucose is glycosidic bonded to the reducing terminal hydroxy group of a maltooligosaccharide having 1 to 10 repeating positions. The glycosidic bond may be either an α-bond or a β-bond. Examples of malto-oligosaccharides include maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and especially maltotetraose, maltopentaose, maltohexaose, and maltopentaose. Preferably 8, the aglycone that binds and separates from malto-oligo circulation in the present invention is released by the action of glucosidase, :S#
Any compound may be used as long as it develops a color or is easy to quantify, but aromatic compounds having a hydroxyl group are particularly preferred.

α− is β− at the reducible end of the uninvented malto-oligo.
In addition to phenol, as aromatic compounds that light the water condition group t° bound by the bond G, there are
Phenols having a roxy group, an alkyl group having 1 to 6 carbon atoms, an alkoxy group, an alkoxycarbonyl substrate fc hanitro group, such as chlorophenol, dichlorophenol, hydroxyphenol, alkylphenol, alkoxyphenol, benzoin or nitrophenol, Examples include halogenated nitrophenol, alkylated nitrophenol, alkoxylated nitrophenol, nitrated benzoic acid, and dinitrophenol.

It may also be an aromatic compound such as 17t 4-methyl-umbelliferone.

In particular phenols having at least one nitro:1 group, such as 4-ditrophenol, 2-chloro-4-nitrophenol, 2,6-dichloro-4-nitrophenol, 2,6-dipromo-4-nitrophenol. Phenol, 2-
7%-4-nitrophenol, 2-nitrophenol, 2-hydroxy-4-nitrophenol, 3-hydroxy-4-nitrophenol, and the like are preferred.

Next, to explain the sounding body of the present invention using four examples,
Examples include the following.

α-72nylpentaoside, α-2-chlorophenylpentaoside, α-2,6-cyclofuzunylbentaoside, α-4-nitrof1nylpentaoside, α
-2-chloro-4°-nitrophenylpentaoside,
These β-conjugates include α-2-methyl-4-nitrophenylpentaoside, etc.几includes tetraoside, hexaoside, and pentaoside. You can also mix it with a mixture of kowara.

For malto-oligos, substituents such as halogens, alkoxycarbonyl groups, aliphatic or aromatic groups are bonded, t-sulfonyl groups, carbonyl groups bonded on aliphatic or aromatic groups,
Maltooligosaccharides with 1,6-linked phenyl groups or haglucose residues may be used, and maltooligosaccharides with f! It also includes partially modified chains, such as those in which two hydroxyl groups are intramolecularly cross-linked.

In misfire +14, glycosides are 0.2 to the total composition
~100 q/r, preferably 0.5~10 q/r, preferably 0.5~10 q/r.

Michaelis constant (Km) of α-amylase for substrate
) value is JI30.3mM, normal)(m3~1
It is 1 to 20 times higher than 0 times the standard value when measured at a pH of 1722 degrees, or 8 degrees during the substrate dissolution to be submitted for inspection and analysis. It is preferable.

The nonionic surfactant used in the VC of the present invention includes:
Anything with an HLB value in the range of 11 to 18 is acceptable, but examples include polyoxyethylene-p-yneoctylphenyl ether, polyoxyethylene lauryl ether, polyoxythere/lauryl ester, and polyoxyethylene-p-nonyl. Phenyl ether, polyoxyethylene cetyl ether, etc.

In the invention, the nonionic surfactant is preferably 0.2 volume fJt% or more based on the total composition, (0.3 to 1.
Contains 2 volumes of rice cake. Normally, in a clinical laboratory test system that measures all body fluids such as blood, lipids and proteins in body fluids: 1
In order to reduce the effects of turbidity caused by
' Less than S. In the present invention, by containing the nonionic surfactant in an amount of 0.2% or more based on the total composition, the glycoside that is the substrate can be stabilized. If the amount is less than 0.2 t% by volume, stabilization cannot be achieved.

In addition to the above glycoside and nonionic surfactant, the composition of the present invention contains a buffer or water.

In the present invention, a nonionic surfactant may be added to the buffer solution or water in which the glycoside is dissolved, or the glycoside may be added to the buffer solution or water in which the nonionic surfactant is dissolved. .

Furthermore, the glycoside and the nonionic surfactant may all be added to the buffer or water at the same time.

The glycoside composition of the present invention may optionally contain a coloring agent such as 4-7 minoantibiline, an enzyme such as α-glucosidase, β-glucosidase, etc., a hydrating agent such as periodic acid, etc. M may be included.

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, a nonionic surfactant is added to the entire composition.
By containing more than 2 volumes, the stability of the array element can be improved. In particular, nonionic surfactants are 0.
．． At 1 t%, stability is lost after storage (1 day at 25°C), but when the nonionic surfactant is at 0.2 volume f# or more, it can be stored stably for 7 days or more.

C implementation column The present invention will be explained below with reference to all embodiments.

Example] Bipes buffer (pH 6,7) 0.05M with β-2,4
-4 μmole of dichlorophenyl maltopentaoside
/- and 4-aminoantipyrine 4 μmole/dt
- In addition, a substrate-containing loose fr'fcf was obtained. Then, 0.6% each of Triton-X (polyoxyethylene octylphenyl ether) or 1.2 volumes of a nonionic surfactant shown in Table 1 was added to prepare a substrate reagent solution.

Obtained nT: In order to measure the stability of the substrate reagent solution, the following enzyme reagent solution and coloring solution were fully opened, and the F absorbance was totally measured in the same manner as above.

Reagent composition ■ Enzyme solution Pipes buffer 0.05 M (pH 6,
7] α-glucosidase 100u/srβ
-Glucosidase 10u/-Triton X
-100 (polyoxyethylene octylphenyl ether) l), 1% ■ Coloring liquid borate buffer 0.1 M (pH 8,4
) Sodium periodate aqueous solution 10 mmol/l sample 2
0 μlt - Transfer to a test tube and mix with 0.5 d of enzyme reagent solution,
After heating at 7℃ for about 5 minutes, add 0.5df of the substrate sample solution, incubate at 37℃ for exactly 10 minutes, add 2.0mt of the coloring solution, and after leaving it for 5 minutes, measure the absorbance at the wavelength. 5
Measured at 00nm, t0 This procedure When using 20μE of water instead of all the samples (reagent blank and 2,4-dichlorophenol 2 mmol/!
When used on an aqueous solution (standard), the measurement was repeated, and the α-amylase value was calculated from the obtained absorbance according to the following formula.

Table 1 shows the changes in the reagent blank when all of the various substrate reagent solutions described above were stored at 4°C and 25°C. The increase in the reagent blank over time is due to the decomposition of the substrate (β-2,4-dichlorophene;
This corresponds to the production of 4-dichlorophenol. Therefore, it is clear that the glycosides in the test solution according to the present invention are stabilized.

I-j・″margin g 1 Table

Claims (1)

[Claims] A stable composition comprising a buffer solution or water containing 0.2 to 100 mg/ml of glycoside based on the total composition and 0.2% by volume or more of a nonionic surfactant based on the total composition. glycoside composition.