JPH0517837B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a reagent used for measuring α-amylase activity in fields such as clinical diagnosis and biochemical research. BACKGROUND TECHNOLOGY Measuring the activity of α-amylase contained in body fluids such as blood and urine provides important basic data in clinical diagnosis and biochemical research.
It is widely practiced. There are various enzyme activity measurement methods that can be used in this case, but one method that has been developed in recent years is to use a substrate or α-amylase in a sample is allowed to act on a substrate consisting of untreated maltooligosaccharides in the presence or absence of α-glucosidase (or α-glucosidase and β-glucosidase), and the liberated indicator compound or glucose is quantified. There is a method for determining the activity of α-amylase (US Pat. No. 3,879,263, Japanese Patent Publication No. 57-53079, etc.). The α-glucosidase and β-glucosidase that are used in this case are called follower enzymes, and when they are used, the hydrolysis of the substrate proceeds stoichiometrically, improving measurement accuracy. Specifically, for example, p-nitrophenyl-α-maltopentaoside as a substrate and α-glucosidase as a follower enzyme are added to a sample containing α-amylase. - Generate p-nitrophenyl-α-maltoside and maltotriose from α-maltopentaoside, then act with a follower enzyme to generate p-nitrophenol and glucose, and generate the former which is an indicator compound. By tracking the temporal changes in the amount α−
Learn about amylase activity. In this case, p-nitrophenol has maximum absorption near a wavelength of 400 nm,
On the other hand, since the substrate to which this is bound, p-nitrophenyl-α-maltopentaocide, has maximum absorption near a wavelength of 300 nm, the free indicator compound can be easily quantified by measuring the absorbance near a wavelength of 400 nm. I can do it. In addition to the above examples, aromatic phenols (hereinafter referred to as nitro group-substituted phenols) have a nitro group directly bonded to the aromatic ring or a nitro group bonded to the aromatic ring via a carbon-carbon double bond conjugated with the aromatic ring. Several methods have been proposed for measuring α-amylase activity using a substrate bound to an indicator compound such as More advantageously, an o-halogen-substituted nitrophenol is bound as an indicator compound.
- There is a method using halogen-substituted nitrophenyl malto-oligoside as a substrate (by the present inventors, patent application filed as Japanese Patent Application No. 111296/1982; unpublished). Problems to be Solved by the Invention The α-amylase activity measurement method using a tracking enzyme is easy to measure and has good measurement accuracy, especially when using a substrate bound to a nitro group-substituted phenol. However, the sample is α-
Even in the case of products that do not contain any amylase, the follower enzyme hydrolyzes the substrate (a so-called blank reaction), giving a false amylase activity measurement.
It is necessary to correct this, and if the blank reaction is rapid, the effective substrate concentration will decrease before the enzyme activity measurement reagent consisting of the substrate and the tracking enzyme is used, and the blank value will also be large. There is a problem that the accuracy of enzyme activity measurement deteriorates over time. The purpose of the present invention is to solve the above-mentioned problems in the α-amylase activity measurement method that uses a substrate and a follower enzyme in combination, and to provide a reagent for α-amylase activity measurement that is difficult to cause a blank reaction and has good stability. There is a particular thing. Means for Solving the Problems The present invention has succeeded in achieving the above objects.
A non-ionic reagent for measuring the activity of α-amylase contains glucoside or maltooligosaccharide formed by bonding a phenol to the reducing molecule end of maltooligosaccharide as a substrate for α-amylase, and α-glucosidase as a follower enzyme. It is characterized by containing a surfactant or dextran sulfate or its salt as an auxiliary agent for suppressing the blank reaction. The amount of the nonionic surfactant, dextran sulfate, or its salt contained in the reagent for measuring α-amylase activity according to the present invention may be about 0.01 to 5 w/v% based on the total liquid volume. For that salt, the lower limit of effective amount is about 0.001%. A nonionic surfactant and dextran sulfate (or its salt) may be used in combination, and in that case, more significant effects can be obtained than when they are used alone. The nonionic surfactant to be included in the reagent for measuring α-amylase activity according to the present invention may be any nonionic surfactant as long as it has an HLB value of 12 or higher without any problems in terms of water solubility, but specific examples of those that can be used are as follows. There is something like. Two or more of these may be used in combination. (i) Polyoxyethylene long-chain alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene oleyl ether, and polyoxyethylene stearyl ether. (ii) Polyoxyethylene alkyl ethers such as polyoxyethylene nonyl phenyl ether and polyoxyethylene octylphenyl ether. (iii) Polyoxyethylene fatty acid esters such as polyoxyethylene monolaurate, polyoxyethylene monooleate, and polyoxyethylene monostearate. (iv) Aliphatic esters of polyoxyethylene alkyl ethers or polyoxyethylene alkylaryl ethers, such as polyoxyethylene stearyl ether laurate and polyoxyethylene nonyl phenyl ether laurate. (v) polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan trilaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, Polyethylene oxide adducts of polyhydric alcohol aliphatic esters, such as polyoxyethylene pentaerythritol monolaurate. (vi) Others include polyethylene glycols, polyoxyethylene-polyoxypropylene block copolymers, polypropylene glycols, etc. Dextran sulfate having a molecular weight of about 50,000 to 1,000,000 is used, and its salt is preferably an alkali metal salt such as sodium salt or potassium salt. The substrate, which is one of the basic components of a reagent for measuring α-amylase activity, is a malto-oligosaccharide or various types of maltooligosaccharides formed from maltooligosaccharides and phenols, which enable the measurement of α-amylase activity according to the principle described above. Although any glucoside can be selected, specific examples of particularly suitable substrates include the following. (i) Maltooligosaccharides maltotetraose, maltopentaose,
Maltohexaose, maltoheptaose, etc. (ii) p-nitrophenyl maltooligosides p-nitrophenyl-α-maltopentaoside, p-nitrophenyl-β-maltopentaoside, p-nitrophenyl-α-maltohexaoside, p-nitrophenyl-β- Maltohexaoside, p-nitrophenyl-α-
Maltoheptaoside, p-nitrophenyl-
β-maltoheptaoside etc. (iii) Halogen-substituted nitrophenyl maltooligosides 2-chloro-4-nitrophenyl-α-
Maltopentaoside, 2-chloro-4-nitrophenyl-β-maltopentaoside, 2-
Chlor-4-nitrophenyl-β-maltohexaoside, 2-chloro-4-nitrophenyl-β-maltoheptaoside, 2,6-dichloro-4-nitrophenyl-α-maltopentaoside, 3,5-dichlor -4-Nitrophenyl-α-maltopentaoside, 2,6-dichloro-4-nitrophenyl-β-maltopentaoside, 2-bromo-4-nitrophenyl-
α-maltopentaoside etc. Further, the follower enzymes, α-glucosidase and β-glucosidase, which are another basic component, may be of any origin, such as α-glucosidase obtained from Satucharomyces carlosbengensis, yeast, etc. Glucosidase and β-glucosidase obtained from almonds can be used. In addition to the above-mentioned components, the reagent for measuring α-amylase activity according to the present invention includes other enzymes necessary for measuring the activity, a buffer solution (preferably one with a pH of 6.5 to 7.5), antibiotics, and sulfur drugs. Chemotherapeutic agents such as, chelating agents (ethylenediaminetetraacetate, etc.), stabilizers (calcium salts, etc.), etc. can be appropriately blended depending on the substrate used and the type of specimen. The reagent for measuring α-amylase activity according to the present invention contains auxiliary agents such as nonionic surfactants that have not been used in the past for measuring α-amylase activity. No special operations are required for the measurement;
- The measurement can be carried out in exactly the same manner as when using a conventional α-amylase activity measuring reagent containing glucosidase as a follower enzyme. That is, α-glucosidase and β
- Glucosidase is added and reacted with a nonionic surfactant or dextran sulfate (or its salt). The activity of α-amylase is then measured by quantifying glucose or phenols released from the substrate using a conventional method. Glucose can be quantified by, for example, using glucose oxidase, reacting the generated hydrogen peroxide with peroxidase and a coloring reagent, and measuring the absorbance of the product. A method for quantifying glucose-6 by an electrical method using a hydrogen electrode
- There is a method of reacting phosphate dehydratase, hexokinase, adenosine-3-phosphate, and nicotinamide adenine dinucleotide and measuring the change in absorbance at 340 nm of the resulting reduced nicotinamide adenine nucleotide. As a method for quantifying phenols, free phenols can be determined, for example, by p-nitrophenol or o-chloro-p.
- In the case of nitrophenol, there is a method to directly measure the change in absorbance.
In the case of 2,6-dichlorophenol, etc., there is a method in which a color reagent such as 4-aminoantipyrine is added, oxidative condensation is carried out, and the absorbance of the product is measured. The reagent of the present invention, like conventional reagents of this type,
It can be widely used to measure the activity of α-amylase contained in serum, urine, pancreatic juice, saliva, etc. Effects of the Invention The reagent for measuring α-amylase activity according to the present invention contains a nonionic surfactant or dextran acid ester or its salt that has the effect of suppressing the blank reaction caused by the follower enzyme as described above. Because of this, it has excellent stability with almost no substrate decomposition before use, and when used to measure α-amylase activity, the blank value is small and there is no interference from added auxiliaries. , it is possible to perform measurements with higher precision than before. EXAMPLES The present invention will be described below with reference to Examples and Experimental Examples. Example 1 50mM PIPES* Buffer (PH7.0) 2-Chlor-4-nitrophenyl-β-maltopentaoside 5mM α-glucosidase 80U/ml β-glucosidase 10U/ml CaCl 1mM NaCl 1mM * Piperazine-N,N '-Bis(2-ethanesulfonic acid) Reagents A to X for measuring the activity of α-amylase were prepared by adding the auxiliary agents listed in Table 1 to the reagent having the above basic composition. In addition, as a control example, a sample (a) without additives was prepared. With the above reagent, serum and urine are used as specimens.
The activity of α-amylase was measured by the method described below. Add 3 ml of the reagent heated at 37°C for 5 minutes to 20μ of the sample to react, and measure the linear portion from 4 minutes to 8 minutes after addition at 400 nm. In order to understand the Frank reaction caused by the following enzyme, similar measurements are performed using water as a sample. Table 1 shows the change in absorbance for 1 minute in each measurement example (measured values for the specimen exclude the measured value when water was used as a sample as a blank value). As is clear from the table, when the Example product of the present invention was used, the blank value decreased by 24% at the lowest value and by 92% at the maximum compared to the control example. Further, no interference by auxiliary agents was observed. (In Table 1 and Table 2 below, polyoxyethylene alkyl ether is manufactured by Nikko Chemicals, BL-8SY, polyoxyethylene alkyl phenyl ether is manufactured by Sigma, Triton (The number is unknown.) Experimental example 50mM PIPES buffer (PH7.0) Maltoheptaose 15mM Glucose-6-phosphate dehydrogenase 6U/ml α-glucosidase 80U/ml Hexokinase 5U/ml CaCl 1mM NaCl 1mM Adenosine 3-phosphate 1mM Nicotinamide adenine dinucleotide 2.5mM Reagents A to X for measuring α-amylase activity were prepared by adding the auxiliary agents listed in Table 2 to the reagent having the above basic composition. In addition, as a control example, a sample (a) without additives was prepared. The absorbance of the above reagent at 340 nm was measured immediately after preparation and after storage at 4°C for 24 hours. The results are shown in Table 2. From the same table, it can be seen that the example products of the present invention show less change in absorbance due to storage than the control example, and have excellent stability.

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Claims (1)

[Claims] 1. A glucoside or maltooligosaccharide formed by bonding a phenol to the terminal end of a reducing molecule of a maltooligosaccharide is contained as a substrate for α-amylase,
- α containing glucosidase as a follower enzyme -
A reagent for measuring α-amylase activity, which contains a nonionic surfactant or dextran sulfate or a salt thereof. 2. The reagent according to claim 1, further containing β-glucosidase as a follower enzyme. 3. The reagent according to claim 1, wherein the substrate is a halogen-substituted nitrophenyl maltooligoside.