WO2018056162A1

WO2018056162A1 - Lipase activity measuremnet method and reagent, and substrate solution for use in measurement of activity of lipase

Info

Publication number: WO2018056162A1
Application number: PCT/JP2017/033234
Authority: WO
Inventors: 直美飯塚; 篤引地
Original assignee: 株式会社シノテスト
Priority date: 2016-09-20
Filing date: 2017-09-14
Publication date: 2018-03-29
Also published as: JPWO2018056162A1; JP6947409B2

Abstract

[Problem] To provide: a substrate solution for use in the measurement of the activity of a lipase, which contains 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin)-ester [DGGMR] as a lipase activity measurement substrate, and in which the deterioration of DGGMR can be prevented and the decrease in an absorbance value obtained by the measurement can be prevented even when the solution is contaminated by an azide; a lipase activity measurement reagent; a lipase activity measurement method; and a method for reducing the influence of an azide. [Solution] A substrate solution for use in the measurement of the activity of a lipase, said substrate solution containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin)-ester [DGGMR] as a lipase activity measurement substrate, wherein a reducing agent is further contained in the substrate solution.

Description

Lipase activity measurement method and reagent, and lipase activity measurement substrate solution

The present invention relates to a lipase activity measurement substrate solution, a lipase activity measurement reagent, and a lipase activity measurement method that can suppress the influence of azide on the lipase activity measurement.
The present invention also relates to a method capable of suppressing the influence of azide on lipase activity measurement.

The present invention is useful in life science fields such as clinical tests and chemical fields such as analytical chemistry.

Since lipase activity in serum or plasma increases in pancreatic diseases such as acute pancreatitis, chronic pancreatitis or pancreatic cancer, it is useful as a marker for these pancreatitis and the like.
This lipase hydrolyzes the ester bond at the α-position (position 1 and position 3) of triglyceride (TG), in which three molecules of long-chain fatty acids are ester-bonded to glycerol, to thereby yield two molecules of fatty acid and one molecule of β-. It is an enzyme that catalyzes a reaction that produces monoglyceride.
This molecule of β-monoglyceride is isomerized into α-form, which is hydrolyzed to glycerol and fatty acid by the action of lipase.

The following methods have been known as methods for measuring lipase activity in serum or plasma (see Non-Patent Document 1 and Non-Patent Document 2).
For example, an olive oil emulsion is used as a lipase substrate, this olive oil emulsion is brought into contact with a serum sample and the like, reacted at 37 ° C. for 24 hours, and then the fatty acid produced by the hydrolysis reaction with lipase is titrated with alkali. Crandall's method was known.
However, this method is a method in which the reaction time is long and the inactivation of the lipase to be measured and the reaction inhibition are remarkable.

In addition, the emulsion of triolein or olive oil is used as a lipase substrate, and the emulsion of triolein or olive oil is brought into contact with a serum sample and reacted to cause turbidity of the reaction solution caused by the hydrolysis reaction of the emulsion micelle by lipase. The Vogel-Zieve method for measuring lipase activity from a decrease in degree and this variant were known.
However, these methods are disadvantageous in that it is difficult to produce a uniform and stable emulsion due to inhibition by serum proteins and interference of aggregates due to rheumatoid factors, and poor reproducibility.

Further, BALB (2,3-dimercapto-1-propanol tributyric acid) is used as a lipase substrate, and this BALB is brought into contact with a serum sample or the like and reacted to produce BAL (2,3 produced by a hydrolysis reaction with lipase). A method for measuring lipase activity by reacting -dimercapto-1-propanol) with DTNB (5,5'-dithiobis-2-nitrobenzoic acid) and measuring the yellow color of the resulting TNB anion at 412 nm. It was known.
However, since this method is subject to interference in the presence of a high concentration of liver esterase, it is affected by the liver esterase mixed in from the measurement reagent of other measurement items via the reaction cell or nozzle (probe). This method has a disadvantage that an error occurs in the measured value.

In addition, 1,2-dilinoleoylglycerol, which is a natural substrate, is used as a lipase substrate, and this 1,2-dilinoleoylglycerol is brought into contact with a serum sample and reacted to produce a lipase hydrolysis reaction. Linoleic acid is a cofactor of acyl-CoA synthetase, acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase-3-ketoacyl-CoA thiolase complex enzyme in the presence of coenzyme A, NAD ⁺ and ATP There has been known a method for measuring lipase activity by measuring the rate of formation of NADH that occurs upon undergoing β-oxidation by action.
However, this method is also subject to interference in the presence of a high concentration of liver esterase, and is therefore affected by liver esterase mixed from the measurement reagent of other measurement items via the reaction cell or nozzle (probe). This method has a disadvantage that an error occurs in the measured value.

In addition to the above measurement methods, 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [hereinafter sometimes referred to as “DGGMR”] was added to lipase A method for measuring lipase activity in serum or plasma used as a substrate has been developed (see Patent Document 1 and Non-Patent Document 2).
In this method, the 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] is brought into contact with a serum sample and reacted, whereby lipase is reacted. The catalyzed hydrolysis reaction produces 1,2-o-dilauryl-rac-glycerol and glutaric acid- (6′-methylresorufin) -ester.
This glutaric acid- (6′-methylresorufin) -ester is unstable and readily hydrolyzes to yield 6′-methylresorufin (λmax: 580 nm).
The increase in 6′-methylresorufin produced is measured by measuring the absorbance at a wavelength of 580 nm or in the vicinity thereof, and the activity value of lipase contained in the sample can be determined.
This method of measuring lipase activity using DGGMR as a lipase substrate is a simple method in which the measurement proceeds in a series of reactions, and the influence of esterase mixed in from other measurement reagents via a reaction cell or nozzle (probe) is considered. This method has the advantage of being difficult to receive.

In addition, lipase contained in serum or plasma acts most efficiently at the water / oil interface of the emulsified triglyceride substrate, and the reaction rate of this lipase is related to the surface area of the dispersed substrate. Preparation of a substrate composed of stable and uniform micelle particles is considered to be important for activity measurement (see Non-Patent Document 2).
Therefore, conventionally, when producing a substrate solution for use in measuring lipase activity (substrate solution for measuring lipase activity), it becomes an emulsified (emulsified) substrate solution composed of stable and uniform micelle particles. Various methods have been considered, but the substrate is mixed with an aqueous solution containing a surfactant, the substrate is mixed with a solution containing an organic solvent such as alcohol, or the substrate-containing solution is dropped dropwise. It is cumbersome or skillful, such as mixing into a solution, spraying and injecting a substrate-containing solution into the solution, stirring the substrate solution at a high speed with a powerful mixer, or applying ultrasonic waves to the substrate solution. Special treatment such as, or the like, or a special device or instrument.

By the way, a solution containing a substrate for measuring lipase activity is generally unstable and has a problem in its storage stability.
For this reason, various attempts have been made for the purpose of improving the storage stability of a solution containing a substrate for measuring lipase activity.

For example, add a non-water-soluble substance such as triglyceride as a lipase substrate to an aqueous solution containing a non-ionic surfactant, heat it with stirring, and once raise it to a temperature higher than the cloud point of the non-ionic surfactant. A method for producing a transparent solubilized aqueous solution of a water-insoluble substance, which is cooled to below the cloud point while continuing to stir, enables the solubilization of a water-insoluble substance, which has been difficult in the past, and is obtained. The solubilized aqueous solution was disclosed as having the effect of being extremely stable (see Patent Document 2).

In addition, a diglyceride solution for measuring lipase activity comprising a low-concentration pH buffer solution and a nonionic surfactant such as a polyoxyethylene alkylphenyl ether-based nonionic surfactant has long-term storage stability. It was disclosed as having an effect that it is possible to provide an aqueous solution of diglyceride having excellent properties (see Patent Document 3).

Also, a composition for measuring plant-derived and / or microorganism-derived lipase activity, comprising using, as a substrate, diglyceride dissolved in at least a nonionic surfactant such as a polyoxyethylene alkylphenyl ether-based nonionic surfactant However, it has been disclosed that the kit and composition containing an aqueous solution of diglyceride excellent in long-term storage stability can be provided (see Patent Document 4).

Further, a lipase substrate solution for enzyme activity measurement, etc. solubilized with 1,2-diphthalanoyl-sn-glycero-3-phosphocholine, a novel lipase substrate solubilizing agent, with respect to the lipase substrate composed of the above DGGMR, It was disclosed that the storage stability is large and that the high transparency of the solution can be maintained over a long period of time (see Patent Document 5).

Further, a mixture is prepared by mixing the lipase substrate composed of the above DGGMR and a side chain type non-reactive polyether-modified type modified silicone oil or polyoxyethylene / polyoxypropylene condensate. It was disclosed as a method for producing a solution for measuring lipase activity, comprising a step of mixing a part with water or an aqueous solution (see Patent Document 6).

Japanese Patent Laid-Open No. 61-254197 JP 58-156330 A International Publication No. 06/054681 pamphlet JP 2007-306721 A JP 11-318494 A International Publication No. 2016/024549 Pamphlet

Reagents are often unstable and often deteriorate during storage and become unusable.
In particular, when a clinical test reagent is used in an automatic analyzer and the reagent must be used in an opened state, the deterioration of the reagent is significant due to various factors caused by the opened state.

For example, when oxygen in the air dissolves in the reagent, components in the reagent may be oxidized and deteriorated.
Further, when carbon dioxide in the air dissolves in the reagent, the pH of the reagent may be lowered and the original function may not be performed.
Furthermore, when carbon dioxide dissolves in the reagent, the test substance (substance to be measured) is inhibited by carbon dioxide, and accurate measurement may not be possible.

When the present inventors set a lipase activity measurement reagent containing DGGMR as a lipase activity measurement substrate in an automatic analyzer and measure the lipase activity value in a sample, the absorbance value obtained by the measurement is changed over time. I noticed that it gradually declined.

As a result of the study, the present inventors have vaporized, as hydrogen azide, azide such as sodium azide contained as a preservative in other clinical test reagents in the vicinity of the reagent container of the automatic analyzer. As a result, the DGGMR contained in the lipase activity measurement substrate solution deteriorates and the absorbance value obtained by measuring the lipase activity value in the sample. Was found to decrease over time.

Furthermore, the inventors of the present invention use an autoanalyzer for measuring lipase activity in which azide contained in a reagent adheres to a reagent probe (reagent sampling port) of the autoanalyzer, and this reagent probe is the next reagent. When a substrate solution (containing DGGMR as a lipase activity measurement substrate) is collected, the azide adhering to the reagent probe is mixed in the lipase activity measurement substrate solution, and the DGGMR contained in the lipase activity measurement substrate solution It was found that the absorbance value obtained by measuring the lipase activity value in the sample was lowered.

As described above, the conventional lipase activity measurement substrate solution containing DGGMR as a lipase activity measurement substrate deteriorates DGGMR by mixing with azide contained in other clinical laboratory reagents, and is obtained by measurement. As a result, the absorbance value was decreased.

On the other hand, the problem of the present invention is to prevent the degradation of DGGMR in a lipase activity measurement substrate solution containing DGGMR as a lipase activity measurement substrate, even if azide is mixed, and to obtain an absorbance value obtained by measurement. It is to provide a lipase activity measurement substrate solution capable of suppressing the decrease.

Another object of the present invention is to prevent degradation of DGGMR and suppress a decrease in absorbance value obtained by measurement even if azide is mixed in a lipase activity measurement reagent containing DGGMR as a lipase activity measurement substrate. It is to provide a reagent for measuring lipase activity capable of

Another object of the present invention is to provide a lipase activity measurement method using a lipase activity measurement substrate solution containing DGGMR as a lipase activity measurement substrate, even if azide is mixed therein, preventing deterioration of DGGMR and obtaining it by measurement. It is providing the lipase activity measuring method which can suppress the fall of the light absorbency value produced.

Furthermore, the object of the present invention is to prevent degradation of DGGMR and suppress a decrease in absorbance value obtained by measurement even when azide is mixed in lipase activity measurement using DGGMR as a substrate for lipase activity measurement. It is providing the suppression method of the influence of the azide which can be performed.

The inventors of the present invention have studied the lipase activity measurement substrate solution, the lipase activity measurement reagent, the lipase activity measurement method, and the method for suppressing the influence of azide, which contain DGGMR as a lipase activity measurement substrate. It has been found that the above problem can be solved by including a reducing agent together with DGGMR in the substrate solution, and the present invention has been completed.

The gist of the present invention is as follows.
(1) Substrate solution for lipase activity measurement containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate, containing a reducing agent A substrate solution for measuring a lipase activity.
(2) A lipase activity measurement reagent comprising the lipase activity measurement substrate solution according to (1).
(3) Lipase activity measurement using a lipase activity measurement substrate solution containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate A method for measuring lipase activity, characterized in that a reducing agent is contained in a substrate solution for measuring lipase activity.
(4) A reducing agent is contained in a lipase activity measurement substrate solution containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate. A method for suppressing the influence of azide on lipase activity measurement.

The substrate solution for measuring lipase activity of the present invention is a substrate solution for measuring lipase activity capable of preventing the degradation of DGGMR due to the mixed azide and suppressing the decrease in the absorbance value obtained by the measurement.
The reagent for measuring lipase activity of the present invention is a reagent for measuring lipase activity capable of preventing the degradation of DGGMR due to a mixed azide and suppressing the decrease in the absorbance value obtained by the measurement.
Moreover, the lipase activity measuring method of the present invention is a lipase activity measuring method capable of preventing the degradation of DGGMR due to the mixed azide and suppressing the decrease in the absorbance value obtained by the measurement.
In addition, the method for suppressing the influence of an azide according to the present invention is a method for suppressing the influence of an azide that can prevent the degradation of DGGMR due to a mixed azide and suppress a decrease in the absorbance value obtained by the measurement.

[1] Substrate solution for measuring lipase activity of the present invention General 1. Substrate solution for lipase activity measurement The substrate solution for lipase activity measurement of the present invention comprises 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] lipase activity. The lipase activity measurement substrate solution contained as the measurement substrate contains a reducing agent.

And the substrate solution for lipase activity measurement of the present invention can prevent the degradation of DGGMR due to the mixed azide and suppress the decrease in the absorbance value obtained by the measurement by the above-mentioned constitution.

2. Lipase In the present invention, the lipase is not particularly limited as long as it has an activity as a lipase, that is, a lipase activity.

In the present invention, as the lipase, for example, two molecules of fatty acid are obtained by hydrolyzing the ester bond at the α-position (position 1 and position 3) of triglyceride (TG) in which three molecules of long-chain fatty acid are ester-bonded to glycerol. And pancreatic lipase [EC 3.1.1.3], which catalyzes a reaction for producing one molecule of β-monoglyceride.

The present invention is suitable for measuring the activity of lipase present in body fluid, organ or tissue, more suitable for measuring the activity of lipase present in body fluid, and more suitable for measuring the activity of lipase present in blood, serum or plasma. And is particularly suitable for measuring the activity of lipase present in serum or plasma.

The present invention is also suitable for measuring pancreatic lipase activity.

II. Substrate solution for lipase activity measurement General As described above, the lipase activity measurement substrate solution of the present invention comprises 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester and a surfactant. It is preferable that the emulsion solution is composed of micelle particles and contains a reducing agent.

In the present invention, an emulsion solution composed of micelle particles composed of DGGMR and a surfactant and containing a reducing agent will be described below.

The emulsion solution in the present invention [that is, an emulsion solution composed of micelle particles composed of DGGMR and a surfactant and containing a reducing agent] is a DGGMR, a surfactant, and a reducing agent as described later. It may contain things other than the agent.

2. Substrate for Measuring Lipase Activity (1) General In the present invention, a lipase substrate for use in measuring the activity of lipase contained in a sample, that is, a substrate for measuring lipase activity is 1,2-o-dilauryl-rac- Glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR].

In the present invention, DGGMR, which is a substrate for measuring lipase activity, is brought into contact with a sample and reacted with the lipase contained in the sample, whereby 1,2-o-dilauryl-- is obtained from DGGMR by a hydrolysis reaction catalyzed by the lipase. rac-glycerol and glutaric acid- (6′-methylresorufin) -ester are formed.
This glutaric acid- (6′-methylresorufin) -ester is unstable and readily hydrolyzes to yield 6′-methylresorufin (λmax: 580 nm).
In the present invention, the increase in 6′-methylresorufin produced can be measured by measuring the absorbance at a wavelength of 580 nm or in the vicinity thereof, and the activity value of lipase contained in the sample can be determined.

DGGMR is commercially available from Roche Diagnostics Inc. [Japan] or Sigma Aldrich Japan LLC [Japan].

(2) Concentration of the substrate for measuring lipase activity The concentration of DGGMR in the substrate solution for measuring lipase activity of the present invention, and the concentration of DGGMR in the substrate solution for measuring lipase activity of the present invention is 0.05 mM or more. The emulsion solution is preferably composed of stable and uniform micelle particles.

The concentration of DGGMR in the lipase activity measurement substrate solution of the present invention is more preferably 0.1 mM or more, particularly preferably 0.2 mM or more, for the purpose described above.

The concentration of DGGMR contained in the lipase activity measurement substrate solution of the present invention is preferably 2 mM or less for the above purpose.

Note that the concentration of DGGMR in the lipase activity measurement substrate solution of the present invention is more preferably 1 mM or less, and particularly preferably 0.8 mM or less, for the purpose described above.

3. Reductant (1) General The lipase activity measurement substrate solution of the present invention is characterized in that the lipase activity measurement substrate solution containing DGGMR as the lipase activity measurement substrate contains a reducing agent.

Thereby, the substrate solution for lipase activity measurement of the present invention can prevent the degradation of DGGMR due to the mixed azide, and can suppress the decrease in the absorbance value obtained by the measurement.
The substrate solution for measuring lipase activity according to the present invention is an emulsion solution composed of micelle particles composed of DGGMR and a surfactant, and preferably contains a reducing agent.

(2) Reducing agent in the present invention The reducing agent in the present invention is not particularly limited as long as it has a reducing ability.

Examples of the reducing agent include hydroxylammonium salts and thiol compounds.

(A) Hydroxyl ammonium salt In the present invention, examples of the hydroxyl ammonium salt include hydroxyl ammonium chloride, hydroxyl ammonium nitrate, and hydroxyl ammonium sulfate.

(B) Thiol Compound In the present invention, examples of the thiol compound include dithiothreitol (DTT), N-acetyl-L-cysteine (NAC), thioglycerol, reduced glutathione, cysteine, dithioerythritol, bromide 2 -SH groups such as aminoethylisothiouronium, 2-thioglucose, thioglycolic acid, 2-mercaptoethanol, N-guanyl-L-cysteine, mercaptoacetic acid, mercaptosuccinic acid, 2-mercaptoethanesulfonic acid, or cysteamine The compound which has can be mentioned.

(3) Concentration The concentration of the reducing agent in the lipase activity measurement substrate solution of the present invention is not particularly limited, but is preferably 0.01 mM or more.

The lower limit of the preferred concentration of the reducing agent is more preferably 0.1 mM or more, and particularly preferably 0.5 mM or more.

The concentration of the reducing agent is not particularly limited, but up to 100 mM is sufficient considering the cost and the like.

The lower limit of the preferred concentration of the reducing agent is more preferably 10 mM or less, and particularly preferably 5 mM or less.

(4) Use of reducing agent, etc. In the present invention, the reducing agent may be one type or a plurality of types.

In the present invention, for the purpose of preventing the degradation of DGGMR due to the mixed azide and suppressing the decrease in the absorbance value obtained by measurement, the reducing agent is preferably a hydroxylammonium salt, particularly preferably hydroxylammonium chloride.

4). Surfactant (1) General As described above, the substrate solution for lipase activity measurement of the present invention is an emulsion solution composed of micelle particles composed of DGGMR and a surfactant, and contains a reducing agent. preferable.

Examples of this surfactant include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants.

Examples of the nonionic surfactant include a side chain type non-reactive polyether-modified type modified silicone oil or a polyoxyethylene / polyoxypropylene condensate.

In the substrate solution for measuring lipase activity of the present invention, the surfactant is preferably a nonionic surfactant.
As the nonionic surfactant, a side chain type non-reactive polyether-modified type modified silicone oil or a polyoxyethylene / polyoxypropylene condensate is preferable. A type of modified silicone oil is particularly preferred.

A substrate solution for measuring lipase activity, which is an emulsion solution composed of micelle particles composed of DGGMR and a surfactant, and contains a reducing agent, that is, an emulsion solution comprising DGGMR, a surfactant, and a reducing agent. It contains an agent.

(2) Side-chain non-reactive polyether-modified type modified silicone oil Side-chain non-reactive polyether-modified type modified silicone oil [hereinafter sometimes referred to as “this modified silicone oil”] This will be described below.

The silicone compound is a polymer compound in which a siloxane bond [—Si—O—Si—] is a main chain and an organic group such as a methyl group [CH ₃ —] is bonded to a silicon atom as a side chain.
The linear silicone compound is silicone oil.

The modified silicone oil is composed of a part of silicon atoms of a linear dimethyl silicone compound [Si (CH ₃ ) ₃ —O— [Si (CH ₃ ) ₂ —O—] m—Si (CH ₃ ) ₃ ]. It is a compound in which an organic group is introduced.
This modified silicone oil includes various kinds of organic compounds on a part of polysiloxane side chain, one end of polysiloxane, both ends of polysiloxane, or part of polysiloxane side chain and both ends. There are silicone oils with introduced groups.

Among these, a silicone oil in which various organic groups are introduced into a part of the side chain of polysiloxane is a side chain type modified silicone oil [Si (CH ₃ ) ₃ —O— [Si (CH ₃ ) ₂ —O—. ] M- [Si (CH ₃ ) (organic group) -O-] n-Si (CH ₃ ) ₃ ].

The modified silicone oil is classified into a reactive silicone oil and a non-reactive silicone oil depending on the nature of the organic group to be introduced.

Among these, as non-reactive modified silicone oil, polyether modified type, aralkyl modified type, fluoroalkyl modified type, long chain alkyl modified type, higher fatty acid ester modified type, higher fatty acid amide modified type, depending on the introduced organic group And polyether / long-chain alkyl / aralkyl-modified type, long-chain alkyl / aralkyl-modified type, and phenyl-modified type.

Then, a side-chain non-reactive modified silicone oil [Si (CH ₃ ) ₃ —O— [Si (CH ₃ ) ₂ —O—] m- [Si (CH ₃ ) (organic group) —O—] n-Si (CH ₃ ) ₃ ] is, for example, a polyether-modified type [organic group: —R (C ₂ H ₄ O) _a (C ₃ H ₆ O) _b R ′] , Polyether, long chain alkyl, aralkyl-modified type [organic group: —R (C ₂ H ₄ O) _a (C ₃ H ₆ O) _b R ′, —C _a H _{2a + 1} , —CH ₂ —CH ( CH ₃ ) —C ₆ H ₅ ], aralkyl-modified type [organic group: —CH ₂ —CH (CH ₃ ) —C ₆ H ₅ ], fluoroalkyl-modified type [organic group: —CH ₂ CH ₂ CF _3], a long-chain alkyl-modified type ones [organic _group: -C _{a H 2a 1],} long-chain alkyl-aralkyl-modified type ones [organic _{_{_{group: -C a H 2a + 1,}}} -CH 2 -CH (CH 3) -C 6 H 5 ], higher fatty acid ester-modified type ones [Organic groups: - OCOR], higher fatty acid amide modified type [organic group: —RNHCOR ′], phenyl modified type [organic group: —C ₆ H ₅ ], and the like.

In the present invention, this side-chain non-reactive polyether-modified type modified silicone oil [Si (CH ₃ ) ₃ —O— [Si (CH ₃ ) ₂ —O—] m- [Si (CH ₃ ) (Organic group) -O-] n-Si (CH ₃ ) ₃ ] [organic group: —R (C ₂ H ₄ O) _a (C ₃ H ₆ O) _b R ′] is preferably used.

As this side chain type non-reactive polyether-modified type modified silicone oil, for example, “KF-351A”, “KF-354L”, “KF-355A”, or “KF-6011” [distributor is Both products are commercially available from Shin-Etsu Chemical Co., Ltd. (Japan).

(3) Polyoxyethylene / polyoxypropylene condensate The polyoxyethylene / polyoxypropylene condensate [hereinafter sometimes referred to as “the present POE / POP condensate”] will be described below.

As described above, polyoxyethylene-polyoxypropylene condensate [HO (C ₂ H ₄ O) _a- (C ₃ H ₆ O) _b- (C ₂ H ₄ O) _c H is used as a nonionic surfactant. Can be mentioned.

As this polyoxyethylene-polyoxypropylene condensate (this POE / POP condensate), for example, polyoxyethylene (16) polyoxypropylene glycol (17) [quasi-drug raw material standard name: polyoxyethylene polyoxy Propylene glycol (16E.O.) (17P.O.)], or polyoxyethylene (20) polyoxypropylene glycol (20) [quasi-drug raw material standard name: polyoxyethylene polyoxypropylene glycol (20E.O.) .) (20P.O.)] and the like.

Examples of the POE / POP condensate include polyoxyethylene (16) polyoxypropylene glycol (17) [product name: “Pluronic L-34”, distributor: ADEKA (Japan)], or poly Oxyethylene (20) polyoxypropylene glycol (20) [Product name: “Pluronic L-44”, distributor: ADEKA Corporation (Japan)], etc. are commercially available.

(4) Concentration of surfactant In the substrate solution for measuring lipase activity, which is an emulsion solution composed of DGGMR and micelle particles made of a surfactant, and containing a reducing agent, the concentration of the surfactant is 0. It is preferable that it is 01% (w / v) or more for preparing an emulsion solution composed of stable and uniform micelle particles.

The concentration of the surfactant in the substrate solution for measuring lipase activity is more preferably 0.05% (w / v) or more, particularly preferably 0.1% (w / v) for the above purpose. v) Above.

The concentration of the surfactant contained in the lipase activity measurement substrate solution is preferably 20% (w / v) or less for the purpose described above.

The concentration of the surfactant in the substrate solution for measuring lipase activity is more preferably 10% (w / v) or less, particularly preferably 5% (w / v) or less, for the above purpose. is there.

5). Lipase Activator (1) Lipase Activator The lipase activator substrate solution of the present invention may contain a lipase activator.

In the present invention, the lipase activator is not particularly limited as long as it is a substance capable of activating lipase, and examples thereof include bile acids or salts thereof.

Examples of the bile acids include deoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, cholic acid, lithocholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, 7-oxolithocholic acid, 12 -Oxolithocholic acid, 12-oxochenodeoxycholic acid, 7-oxodeoxycholic acid, hyocholic acid, hyodeoxycholic acid, dehydrocholic acid, or cholic acid derivatives.

Examples of the bile acid salt include salts of bile acids and alkali metals or alkaline earth metals, ammonium salts, and the like.
Examples of the alkali metal include potassium, sodium, and lithium, and examples of the alkaline earth metal include magnesium and calcium.

As the lipase activator, bile acid or a salt thereof is preferable from the viewpoint of the ability to activate the lipase, the ability to form an interface composed of a substrate for measuring lipase activity, water solubility, and cost.

As the bile acid, taurodeoxycholic acid is preferable in that DGGMR as a lipase activity measurement substrate can be dissolved in a stable acidic region, and deoxycholic acid is preferable from the viewpoint of cost.
As this bile acid, taurodeoxycholic acid is particularly preferable.

The bile acid salt is preferably a bile acid and alkali metal salt, more preferably a bile acid potassium salt or sodium salt, and particularly preferably a bile acid sodium salt.

Therefore, the salt of bile acid is preferably a salt of an alkali metal (such as potassium or sodium) of deoxycholic acid or taurodeoxycholic acid, more preferably a salt of an alkali metal (such as potassium or sodium) of taurodeoxycholic acid, The sodium salt of taurodeoxycholic acid is particularly preferred.

(2) Concentration of lipase activator In the lipase activity measurement substrate solution of the present invention, the lipase activator preferably has a concentration of 0.2% (w / v) or more.

In the lipase activity measuring substrate solution of the present invention, the preferred concentration of the lipase activator is more preferably 0.4% (w / v) or more, particularly preferably 1% (w / v) or more. It is.

The concentration of the lipase activator is preferably 20% (w / v) or less in the lipase activity measurement substrate solution of the present invention.

In the lipase activity measurement substrate solution of the present invention, the preferred concentration of the lipase activator is more preferably 10% (w / v) or less, particularly preferably 5% (w / v) or less. .

6). Lipase Activator (1) Lipase Activator The lipase activity measuring substrate solution of the present invention may contain a lipase activator.

In the present invention, the lipase activator is not particularly limited as long as it is a substance capable of activating lipase, and examples thereof include alkaline earth metal ions or salts thereof.

Examples of the alkaline earth metal ion or salt thereof include beryllium ion or beryllium salt, magnesium ion or magnesium salt, or calcium ion or calcium salt.

Examples of the calcium salt include a water-soluble calcium salt, and more specifically, a salt made of a monovalent or divalent anion and calcium ion and water-soluble. Can be mentioned.

Examples of the anion include halogen ions, acid groups made of organic compounds, and acid groups made of other inorganic compounds.

And as a halogen ion, a fluorine ion, a chlorine ion, etc. can be mentioned, for example.

Examples of the acid group made of an organic compound include acetate ion, citrate ion, or gluconate ion.

Examples of acid groups made of other inorganic compounds include sulfate ions, phosphate ions, or carbonate ions.

The lipase activator is preferably an alkaline earth metal ion or a salt thereof.

In addition, as an alkaline-earth metal ion or its salt, a calcium ion or a calcium salt is preferable from the point of following (i) and (ii).
(I) The ability to activate lipase.
(Ii) Fatty acid liberated from the lipase activity measurement substrate by being catalyzed by lipase breaks the interface comprising the lipase activity measurement substrate, but calcium ions or calcium salts capture the liberated fatty acid, and the interface Can be prevented from breaking.

And as this calcium salt, the salt which consists of an anion and calcium ion and is water-soluble is preferable.

And as this anion, the acid group which consists of a halogen ion or an organic compound is preferable. More specifically, chlorine ion or acetate ion is particularly preferable.

Therefore, as the calcium salt, a calcium salt of a halogen ion or an acid group consisting of an organic compound is preferable, and more specifically, calcium chloride or calcium acetate is particularly preferable.

(2) Concentration of lipase activator In the lipase activity measurement substrate solution of the present invention, the lipase activator preferably has a concentration of 0.1 mM or more.

In the lipase activity measurement substrate solution of the present invention, the preferred concentration of the lipase activator is more preferably 1 mM or more, and particularly preferably 5 mM or more.

The concentration of the lipase activator is preferably 100 mM or less in the lipase activity measurement substrate solution of the present invention.

In the lipase activity measurement substrate solution of the present invention, the preferred concentration of this lipase activator is more preferably 50 mM or less, and particularly preferably 25 mM or less.

7). Colipase (1) Colipase The substrate solution for measuring lipase activity of the present invention may contain colipase.

In the present invention, the colipase is not particularly limited as long as it has the action, function, or activity of colipase. For example, human or a mammal-derived colipase such as pig or genetic engineering is used. And colipase prepared, modified or modified.

As this colipase, a colipase derived from a mammal such as a pig is preferable, and a colipase derived from a pancreas of a mammal such as a pig is more preferable.

(2) Activity value of colipase In the lipase activity measurement substrate solution of the present invention, the activity value of the colipase is preferably 15K units / L (15K Unit / L) or more.

In the lipase activity measurement substrate solution of the present invention, the preferable activity value of this colipase is more preferably 150 K units / L or more, and particularly preferably 750 K units / L or more.

The activity value of this colipase is preferably 7,500 K units / L or less in the lipase activity measurement substrate solution of the present invention.

In the lipase activity measurement substrate solution of the present invention, the preferred activity value of this colipase is more preferably 3,750 K units / L or less, and particularly preferably 2,250 K units / L or less.

In addition, each activity value (unit / L) of colipase in this specification is based on the display of the activity value of colipase derived from porcine pancreas of Roche Diagnostics Inc. [Japan]. [1 mg / L = 75K units / L]

Colipase is commercially available from Roche Diagnostics Inc. [Japan] or Sigma Aldrich Japan LLC [Japan].

8). Water The substrate solution for measuring lipase activity of the present invention may contain water.
That is, the lipase activity measurement substrate solution of the present invention may be an aqueous solution or an aqueous suspension.

The water to be contained in the lipase activity measurement substrate solution of the present invention is not particularly limited, and examples thereof include pure water, distilled water, and purified water.

9. pH
DGGMR as a substrate for measuring lipase activity in the present invention is stable at pH 4 or in the vicinity thereof.
Therefore, in the substrate solution for measuring lipase activity of the present invention, the pH is preferably within a certain range centered on pH 4.

Specifically, the substrate solution for measuring lipase activity of the present invention is preferably in the range of pH 2 to pH 7, more preferably in the range of pH 3 to pH 5, from the viewpoint of the stability of DGGMR. Particularly preferably, the pH is in the range of 3.5 to 4.5. (The above pH values are all values at 20 ° C.)

10. Buffering agent (1) Buffering agent The substrate solution for lipase activity measurement of the present invention may contain a buffering agent.

In the present invention, in order to maintain the pH of the substrate solution for measuring lipase activity of the present invention within the pH range described in 9, the buffer having a buffer capacity in the pH range described in 9 is used for measuring the lipase activity of the present invention. You may make it contain suitably in a substrate solution.

The buffer that can be contained in the lipase activity measurement substrate solution of the present invention is not particularly limited, and examples thereof include organic acids such as tartaric acid, succinic acid, malonic acid, and citric acid, or glycine or phosphoric acid. Or these salts etc. can be mentioned.

(2) Concentration of buffering agent In the present invention, the concentration of the buffering agent contained in the lipase activity measurement substrate solution of the present invention is not particularly limited, and can exhibit a buffering ability within a set pH range. Any concentration is acceptable.

For example, in the lipase activity measurement substrate solution of the present invention, the concentration of this buffer is preferably 5 mM or more, more preferably 10 mM or more, and particularly preferably 30 mM or more.

Further, the concentration of the buffer is preferably 500 mM or less, more preferably 100 mM or less, and particularly preferably 50 mM or less in the substrate solution for measuring lipase activity of the present invention.

11. Micelle size of emulsion of substrate solution for measuring lipase activity As described above, lipase works most efficiently at the water-oil interface of the emulsified triglyceride substrate, and the reaction rate of this lipase is determined by the surface area of the dispersed substrate. Therefore, it is said that the preparation of a substrate composed of stable and uniform micelle particles is important for measuring the activity of lipase (see Non-Patent Document 2).

In the present invention, when the lipase activity measurement substrate solution containing the lipase activity measurement substrate [DGGMR] has a micelle diameter (micelle particle diameter) in the range of 60 to 1,500 nm, the reaction with the lipase The speed is high, the emulsion is stable, and the substrate solution for measuring lipase activity can be stored and used for a long period of time.

For this reason, the lipase activity measurement substrate solution preferably has a micelle diameter (micelle particle diameter) of the emulsion in the range of 70 to 1,000 nm, more preferably in the range of 80 to 600 nm, and The range of 100 to 200 nm is particularly preferable.

III. 1. Method for producing lipase activity measurement substrate solution General description The method for producing a lipase activity measurement substrate solution of the present invention is characterized in that the above-mentioned “lipase activity measurement substrate solution containing DGGMR as a lipase activity measurement substrate contains a reducing agent. There is no particular limitation as long as it is a method capable of producing the lipase activity measurement substrate solution of the present invention having the configuration of “measurement substrate solution”.

In addition, as a method of manufacturing the lipase activity measurement substrate solution of this invention, the method etc. which consist of the process of following (a) and (b) can be mentioned, for example. In the present invention, the “method comprising the following steps (a) and (b)” means “including a method comprising the following steps (a) and (b)”. is there.
The method comprising the steps (a) and (b) is for measuring the lipase activity of the present invention without requiring special treatment such as complicated or skillful need, or special equipment or instruments. Since a substrate solution can be manufactured, it is preferable.

(A) Step of preparing a mixture by mixing DGGMR and surfactant

(B) A step of mixing all or part of the mixture of (a) with water or an aqueous solution.

2. Step of Mixing DGGMR and Surfactant to Prepare Mixture The step (a) in 1 above, that is, “step of mixing DGGMR and surfactant to prepare a mixture” will be described in detail below.

(1) Mixing of DGGMR and Surfactant In the step (a) of 1 above, that is, “the step of preparing a mixture by mixing DGGMR and a surfactant”, DGGMR which is a substrate for measuring lipase activity and the interface Mix the active agent.
That is, DGGMR and the surfactant are directly mixed.

Note that one type of surfactant may be mixed with DGGMR, or a plurality of types of surfactants may be mixed with DGGMR.

(2) Mixing amount of DGGMR In the step of preparing a mixture by mixing DGGMR as a substrate for measuring lipase activity and a surfactant, the amount of mixing DGGMR is not particularly limited.

In addition, this DGGMR is the “DGGMR / surfactant mixture of the DGGMR / surfactant mixture” in the step (b) of “the step of mixing all or part of the DGGMR / surfactant mixture with water or an aqueous solution”. After mixing with “all or part” and the “water or aqueous solution” (hereinafter sometimes referred to as “second mixing”), the concentration is 0.05 mM or more from stable and uniform micelle particles. This is preferable for the purpose of producing an emulsion solution.

In addition, after this 2nd mixing (after 2nd mixing), the preferable density | concentration of this DGGMR is more preferably 0.1 mM or more from the said objective, Especially preferably, it is 0.2 mM or more.

Further, the concentration of DGGMR is preferably 2 mM or less after the second mixing for the above purpose.

In addition, after the second mixing, the preferable concentration of DGGMR is more preferably 1 mM or less, and particularly preferably 0.8 mM or less, for the purpose described above.

The preferred concentration of DGGMR after the second mixing is as described above.
The mixing of the DGGMR and the surfactant in the “step of preparing a mixture by mixing DGGMR and a surfactant” in 1) (a) (hereinafter sometimes referred to as “first mixing”) The mixing amount of each of the DGGMR and the surfactant at the time may be determined in consideration so that the concentration of DGGMR is as described above after the second mixing. Preferred from above.

In addition, although it is the mixing amount and density | concentration of this DGGMR, it can be considered like the following (a) or (b), for example.

(A) When all of the mixture of the lipase activity measurement substrate and the surfactant is mixed with water or an aqueous solution, the mixing amount of the lipase activity measurement substrate mixed during the first mixing is Ws [unit: grams], and the second mixing Sometimes the final volume after mixing water or aqueous solution (volume after volume up, etc.) is Vf [unit: mL] and the molecular weight of the lipase activity measurement substrate is MWs, the lipase after the second mixing The concentration Cs [unit: mM] of the substrate for activity measurement can be expressed by the following formula.

Cs = (Ws × 10 ⁶ ) ÷ (Vf × MWs)

Since the molecular weight MWs of DGGMR, which is a substrate for measuring lipase activity, is 752.05, the above formula is as follows.

Cs = (Ws × 10 ⁶ ) ÷ (Vf × 752.05)

Therefore, the mixing amount Ws [unit: grams] of the lipase activity measurement substrate (DGGMR) to be mixed during the first mixing in this case can be expressed as follows.

Ws = (Cs × Vf × MWs) ÷ 10 ⁶

That is, Ws = (Cs × Vf × 752.05) ÷ 10 ⁶

(B) When a part of the mixture of the lipase activity measurement substrate and the surfactant is mixed with water or an aqueous solution, the mixing amount of the lipase activity measurement substrate mixed at the first mixing is Ws [unit: grams], and the second The final volume after mixing water or aqueous solution at the time of mixing (volume after volume-up, etc.) is Vf [unit: mL], the molecular weight of the lipase activity measurement substrate is MWs, and the mixture of the mixture at the first mixing When A% (weight or volume) is mixed with water or an aqueous solution during the second mixing, the concentration Cs [unit: mM] of the lipase activity measurement substrate after the second mixing can be expressed by the following formula.

Cs = (Ws × 10 ⁶ ) × (A ÷ 100) ÷ (Vf × MWs) = (Ws × A × 10 ⁴ ) ÷ (Vf × MWs)

Cs = (Ws × A × 10 ⁴ ) ÷ (Vf × 752.05)

Therefore, the mixing amount Ws [unit: grams] of the lipase activity measurement substrate to be mixed in the first mixing in this case can be expressed as follows.

Ws = (Cs × Vf × MWs) ÷ (A × 10 ⁴ )

That is, Ws = (Cs × Vf × 752.05) ÷ (A × 10 ⁴ )

(3) Mixing amount of surfactant In the step of preparing a mixture by mixing DGGMR as a lipase activity measurement substrate and a surfactant, the amount of mixing the surfactant is not particularly limited.

The surfactant is preferably at a concentration of 0.01% (w / v) or more after the second mixing for the purpose of producing an emulsion solution composed of stable and uniform micelle particles.

In addition, after the second mixing (after the second mixing), the preferable concentration of the surfactant is more preferably 0.05% (w / v) or more, and particularly preferably 0 for the above purpose. .1% (w / v) or more.

Also, this concentration is preferably 20% (w / v) or less after the second mixing for the above purpose.

In addition, after the second mixing, the preferable concentration of this surfactant is more preferably 10% (w / v) or less, and particularly preferably 5% (w / v) or less, for the above purpose. .

The preferable concentration of the surfactant after the second mixing is as described above.

The mixing amount of the DGGMR and the surfactant during the first mixing may be determined in consideration of the concentration of the surfactant as described above after the second mixing. It is preferable from the viewpoint of the manufacturing procedure.

In addition, although it is the mixing amount and density | concentration of this surfactant, it can be considered like the following (a) or (b), for example.

(A) In the case where the entire mixture of the lipase activity measurement substrate and the surfactant is mixed with water or an aqueous solution, the amount of the surfactant to be mixed during the first mixing is Wp [unit: grams], and the water is mixed during the second mixing. Alternatively, when the final volume after mixing the aqueous solution (volume after volume-up, etc.) is Vf [unit: mL], the concentration Cp [unit:% (w / v) of the surfactant after the second mixing ] Can be expressed by the following equation.

Cp = (Wp × 100) ÷ Vf

Therefore, the mixing amount Wp [unit: grams] of the surfactant to be mixed during the first mixing in this case can be expressed as follows.

Wp = (Cp × Vf) ÷ 100

(B) When a part of the mixture of the lipase activity measurement substrate and the surfactant is mixed with water or an aqueous solution, the amount of the surfactant to be mixed at the first mixing is Wp [unit: grams], and at the second mixing The final volume after mixing water or aqueous solution (volume after measuring up, etc.) is Vf [unit: mL], and A% (weight or volume) of the mixture at the first mixing is water at the second mixing. Alternatively, when mixed with an aqueous solution, the concentration Cp [unit:% (w / v)] of the surfactant after the second mixing can be expressed by the following formula.

Cp = (Wp × 100) × (A ÷ 100) ÷ Vf = (Wp × A) ÷ Vf

Therefore, the mixing amount Wp (unit: gram) of the surfactant to be mixed during the first mixing in this case can be expressed as follows.

Wp = (Cp × Vf) ÷ A

(4) Method of mixing In the step of preparing a mixture by mixing DGGMR and surfactant, the method of mixing DGGMR and surfactant is any method as long as this DGGMR and surfactant are mixed. Well, there is no particular limitation.

In this mixing, DGGMR is mixed with a solution containing an organic solvent such as alcohol and a surfactant, or a solution containing DGGMR is dropped and mixed with a solution containing a surfactant, or DGGMR is mixed. Injecting the liquid containing the surfactant into the solution containing the surfactant, stirring the solution containing the DGGMR and the surfactant at high speed with a powerful mixer, or ultrasonicating the solution containing the DGGMR and the surfactant There is no need for special processing or special equipment, such as processing that requires a lot of time or skill, such as stirring at a general speed using a general mixer. Thus, a mixture of DGGMR and a surfactant can be prepared.

(5) Temperature at the time of mixing In the step of preparing a mixture by mixing DGGMR and a surfactant, the temperature at which the DGGMR and the surfactant are mixed is not particularly limited, but is near the cloud point of the surfactant used. It is preferable for the purpose of producing an emulsion solution composed of stable and uniform micelle particles to carry out this step at a temperature equal to or lower than the temperature near the cloud point.

The cloud point is a temperature at which micelles such as a surfactant cannot be formed when the temperature of an aqueous solution such as a nonionic surfactant is raised, and the aqueous solution becomes cloudy, It is different for each surfactant.

In the present invention, the temperature in the vicinity of the cloud point of the surfactant means a range of plus or minus (±) 25 ° C. of the cloud point temperature of the surfactant.
The temperature near the cloud point of this surfactant is preferably in the range of plus or minus (±) 15 ° C., more preferably in the range of plus or minus (±) 10 ° C., plus or minus. A range of (±) 5 ° C. is particularly preferable.

And in this invention, it is also preferable to perform the process of mixing DGGMR and surfactant and preparing a mixture at the temperature below the cloud point of said surfactant.

For example, the surfactant, KF-351A, has a cloud point of 52 ° C. (self-measured value), KF-355A has a cloud point of 67 ° C. (self-measured value), and KF-6011 The cloud point is 64 ° C. (self-measured value). KF-354L did not reach the cloud point even at 77 ° C. which is the upper limit temperature of the constant temperature water tank used for cloud point measurement. Is above 77 ° C.

The step of mixing the DGGMR and the surfactant to prepare a mixture is performed for the purpose described above, in the range of plus or minus 25 ° C. of the cloud point temperature of the surfactant to be used, or below this range. It is preferable to carry out at a temperature in the range of plus or minus 15 ° C of the cloud point temperature of the surfactant to be used, or more preferably at a temperature below this range. More preferably, it is carried out at a temperature in the range of minus 10 ° C. or below this range, and the temperature of the cloud point of the surfactant used is plus or minus 5 ° C. or below this range. It is particularly preferable to carry out at

Further, in the step of mixing the DGGMR and the surfactant in the present invention to prepare a mixture, this is the temperature at which the DGGMR and the surfactant are mixed. This step is the same as that for the DGGMR and the surfactant to be used. It is preferable to carry out at a temperature above the melting point for the purpose of producing an emulsion solution composed of stable and uniform micelle particles.

The step of mixing the DGGMR and the surfactant to prepare the mixture is preferably performed at 2 ° C. or higher, more preferably at 5 ° C. or higher for the above purpose, more preferably at 10 ° C. or higher. It is particularly preferred.

(6) Mixing time In the step of preparing a mixture by mixing DGGMR and a surfactant, this DGGMR and the surfactant are mixed. If the DGGMR and the surfactant are mixed homogeneously, Well, not particularly limited.
Usually, for the purpose of producing an emulsion solution composed of stable and uniform micelle particles, it is preferable to perform this mixing for 5 minutes or longer. In general, 5 minutes is sufficient.

The time for mixing DGGMR and the surfactant is not particularly limited. For example, it may be mixed for several hours, but from the viewpoint that the time is also cost, even if it is performed carefully, it is usually performed. Within 10 minutes.

3. Step of mixing a mixture of DGGMR and a surfactant with water or an aqueous solution Step (b) in 1 above, that is, all of the mixture prepared in “Step of mixing DGGMR and a surfactant to prepare a mixture” or Hereinafter, the “part of mixing with water or an aqueous solution” will be described in detail.

(1) Water or aqueous solution In the step (b) of 1 above, that is, “the step of mixing all or part of the mixture prepared in the step (a) of 1 with water or an aqueous solution”, There is no particular limitation on the aqueous solution.

The water is not particularly limited, and examples thereof include pure water, distilled water, and purified water.

The aqueous solution is not particularly limited as long as water is used as a solvent. For example, the aqueous solution is selected from the group consisting of a reducing agent, a lipase activator, a lipase activator, a colipase, and a buffer. Examples thereof include an aqueous solution containing at least one.

(A) Reducing agent In the present invention, the reducing agent that can be contained in the aqueous solution is not particularly limited as long as it has a reducing ability, and examples thereof include hydroxylammonium salts and thiol compounds. be able to.

This reducing agent is as described in the section of “3. Reducing agent” in II above.

The concentration of the reducing agent is preferably 0.01 mM or more after the second mixing.

In addition, after the second mixing, the preferable concentration of the reducing agent is more preferably 0.1 mM or more, and particularly preferably 0.5 mM or more.

The concentration of the reducing agent is preferably 100 mM or less after the second mixing.

Note that after the second mixing, the preferred concentration of the reducing agent is more preferably 10 mM or less, and particularly preferably 5 mM or less.

The preferable concentration of the reducing agent after the second mixing is as described above.
In consideration of the mixing ratio of the above-mentioned “mixture of DGGMR and surfactant” and the aqueous solution so that the concentration of the reducing agent after the second mixing becomes the above concentration, an appropriate reducing agent is added to the aqueous solution. It is preferable to contain by concentration.

(B) Lipase activator In the present invention, the lipase activator that can be contained in the aqueous solution is not particularly limited as long as it is a substance that can activate lipase. For example, bile An acid or its salt can be mentioned.

The bile acid or salt thereof as the lipase activator is as described in the section “(1) Lipase activator” in 5 of II.

In addition, it is preferable that the density | concentration of this lipase activator is 0.2% (w / v) or more after 2nd mixing.

In addition, after the second mixing, the preferable concentration of the lipase activator is more preferably 0.4% (w / v) or more, and particularly preferably 1% (w / v) or more.

The concentration of the lipase activator is preferably 20% (w / v) or less after the second mixing.

Note that, after the second mixing, the preferable concentration of the lipase activator is more preferably 10% (w / v) or less, and particularly preferably 5% (w / v) or less.

The preferred concentration of the lipase activator after the second mixing is as described above.
In consideration of the mixing ratio of the above-mentioned “DGGMR and surfactant mixture” and the aqueous solution so that the concentration of the lipase activator after the second mixing is the above-mentioned concentration, the aqueous solution is activated with lipase. It is preferable to contain the agent in an appropriate concentration.

(C) Lipase activator In the present invention, the lipase activator that can be contained in the aqueous solution is not particularly limited as long as it is a substance that can activate lipase. Examples include earth metal ions or salts thereof.

The alkaline earth metal ion or salt thereof as the lipase activator is as described in the section “(1) Lipase activator” in 6 of II.

The lipase activator preferably has a concentration of 0.1 mM or more after the second mixing.

In addition, the preferable concentration of this lipase activator after the second mixing is more preferably 1 mM or more, and particularly preferably 5 mM or more.

The concentration of the lipase activator is preferably 100 mM or less after the second mixing.

Note that, after the second mixing, the preferred concentration of the lipase activator is more preferably 50 mM or less, and particularly preferably 25 mM or less.

The preferable concentration of the lipase activator after the second mixing is as described above.
In consideration of the mixing ratio of the above-mentioned “DGGMR and surfactant mixture” and the aqueous solution so that the concentration of the lipase activator after the second mixing becomes the above-mentioned concentration, the aqueous solution is activated with lipase. It is preferable to contain the agent in an appropriate concentration.

(D) Colipase In the present invention, the colipase that can be contained in the aqueous solution is not particularly limited as long as it has the action, function, or activity of colipase.

This colipase is as described in the section “(1) Colipase” in 7 of II above.

In addition, it is preferable that the activity value of this colipase is 15K unit / L (15K Unit / L) or more after the second mixing.

In addition, the preferable activity value of this colipase after the second mixing is more preferably 150 K units / L or more, and particularly preferably 750 K units / L or more.

The activity value of this colipase is preferably 7,500 K units / L or less after the second mixing.

In addition, the preferable activity value of this colipase after the second mixing is more preferably 3,750 K units / L or less, and particularly preferably 2,250 K units / L or less.

The preferable activity value of the colipase after the second mixing is as described above.
In consideration of the mixing ratio of the above-mentioned “mixture of DGGMR and surfactant” and the aqueous solution so that the activity value of the colipase after the second mixing becomes the above-mentioned activity value, an appropriate amount of colipase is added to the aqueous solution. It is preferable to contain by activity value.

(E) pH
In the present invention, DGGMR used as a substrate for measuring lipase activity is stable at pH 4 or in the vicinity thereof.
Therefore, after the second mixing, the pH is preferably within a certain range centered on pH 4.

Specifically, the pH after the second mixing is preferably in the range of pH 2 to pH 7, more preferably in the range of pH 3 to pH 5, from the viewpoint of the stability of DGGMR, and pH 3. A range of 5 to 4.5 is particularly preferable. (The above pH values are all values at 20 ° C.)

The pH after the second mixing is as described above.
It is preferable to adjust the pH of the aqueous solution to an appropriate pH so that the pH after the second mixing becomes the above pH.

(F) Buffering agent In the present invention, in order to maintain the pH after the second mixing in the pH range described in (e) above, a buffering agent having a buffering ability in the pH range of (e) is appropriately added to the aqueous solution. You may make it contain.

This buffering agent is as described in the section “(1) Buffering agent” in 10 of II above.

The concentration of the buffering agent in the aqueous solution containing this buffering agent (that is, the buffering solution) is not particularly limited as long as the buffering ability can be exhibited in the set pH range.

For example, after the second mixing, the concentration of the buffer is preferably 5 mM or more, more preferably 10 mM or more, and particularly preferably 30 mM or more.

Further, the concentration of this buffering agent is preferably 500 mM or less, more preferably 100 mM or less, and particularly preferably 50 mM or less after the second mixing.

The preferable concentration of the buffering agent after the second mixing is as described above.
It is preferable to contain the buffering agent at an appropriate concentration in the aqueous solution so that the concentration of the buffering agent after the second mixing becomes the above-mentioned concentration.

(2) Mixing of DGGMR and Surfactant Mixture with Water or Aqueous Solution Step (b) in 1 above, ie, the “prepared mixture of DGGMR and surfactant to prepare mixture” In the step of mixing all or a part with water or an aqueous solution, all or a part of the “mixture of DGGMR and surfactant” is mixed with the “water or aqueous solution”.

By the way, it is a step of mixing all or part of a mixture prepared by mixing DGGMR as a lipase activity measurement substrate and a surfactant with water or an aqueous solution, but there is no particular limitation. For example, “DGGMR and interface A mode in which all or part of the “mixture prepared by mixing the active agent” is added to “water or aqueous solution” and mixed, or “water or aqueous solution” is prepared by mixing “DGGMR and surfactant”. The mixture may be added to all or a part of the “mixed mixture” and mixed, or other embodiments may be employed.

The mixing ratio of the “DGGMR / surfactant mixture” and the “water or aqueous solution” is not particularly limited and may be determined as appropriate.

The mixing of the “DGGMR / surfactant mixture” and “water or aqueous solution” can be considered, for example, as in the following (i) and (ii).

(I) From the aspect of DGGMR concentration As described in detail in section 2 “(2) Mixing amount of DGGMR”, after the second mixing, the preferable concentration of DGGMR is preferably 0 for the above purpose. 0.05 mM or more, more preferably 0.1 mM or more, and particularly preferably 0.2 mM or more.
Also, as described in detail in the section “(2) DGGMR mixing amount” in 2 above, the preferable concentration of DGGMR after the second mixing is preferably 2 mM or less for the above purpose. It is preferably 1 mM or less, and particularly preferably 0.8 mM or less.

The relationship between the preferable concentration of DGGMR and the final volume after mixing water or an aqueous solution during the second mixing can be considered as, for example, the following (a) or (b).

Cs = (Ws × 10 ⁶ ) ÷ (Vf × MWs)

Cs = (Ws × 10 ⁶ ) ÷ (Vf × 752.05)

Therefore, the final volume Vf [unit: mL] after mixing water or an aqueous solution in the second mixing in this case can be expressed as follows.

Vf = (Ws × 10 ⁶ ) ÷ (Cs × MWs)

That is, Vf = (Ws × 10 ⁶ ) ÷ (Cs × 752.05)

Therefore, by mixing water or an aqueous solution during the second mixing so that the volume Vf [unit: mL] obtained by the above formula is obtained, the lipase activity measurement substrate having the desired lipase activity measurement substrate (DGGMR) concentration is obtained. A solution can be obtained.

Cs = (Ws × A × 10 ⁴ ) ÷ (Vf × 752.05)

Vf = (Ws × A × 10 ⁴ ) ÷ (Cs × MWs)

That is, Vf = (Ws × A × 10 ⁴ ) ÷ (Cs × 752.05)

(Ii) From the viewpoint of the surfactant concentration As described in detail in the section “(3) Mixing amount of surfactant” in 2 above, the preferable concentration of the surfactant after the second mixing is the above-mentioned purpose. From the top, it is preferably 0.01% (w / v) or more, more preferably 0.05% (w / v) or more, and particularly preferably 0.1% (w / v) or more. is there.

Also, as described in detail in the section “(3) Amount of surfactant” in 2 above, the preferable concentration of the surfactant after the second mixing is preferably 20% for the above purpose. (W / v) or less, more preferably 10% (w / v) or less, and particularly preferably 5% (w / v) or less.

The relationship between the preferable concentration of the surfactant and the final volume after mixing water or an aqueous solution during the second mixing can be considered as, for example, (a) or (b) below. it can.

Cp = (Wp × 100) ÷ Vf

Vf = (Wp × 100) ÷ Cp

Therefore, by mixing water or an aqueous solution at the time of the second mixing so that the volume Vf [unit: mL] obtained by the above formula is obtained, a substrate solution for lipase activity measurement having a desired surfactant concentration can be obtained. it can.

Cp = (Wp × 100) × (A ÷ 100) ÷ Vf = (Wp × A) ÷ Vf

Vf = (Wp × A) ÷ Cp

The process of mixing all or part of the mixture prepared by mixing DGGMR as a lipase activity measurement substrate and a surfactant with water or an aqueous solution is performed in a plurality of stages (steps). May be.
In this way, it is preferable to carry out this step in a plurality of steps for the purpose of producing an emulsion solution composed of stable and uniform micelle particles.

Although this method is a method of performing a plurality of steps, this is not particularly limited as long as the step is performed by a plurality of steps. For example, it is performed by the following steps [A] and [B]. The thing etc. can be mentioned.

[A] Step of mixing all or part of the mixture prepared by mixing DGGMR and surfactant with a certain amount of water or aqueous solution [B] The mixture (DGGMR and surfactant in the step [A] A certain amount of water or aqueous solution is further mixed with the mixed solution of the mixture of water and aqueous solution.

In this case, the volume (constant amount) of “water or aqueous solution” to be mixed with “all or part of the mixture prepared by mixing DGGMR and surfactant” in the step [A] is Va [unit: mL], and the final volume after mixing “more fixed amount of water or aqueous solution” with the mixture in the step [B] [ie, after mixing water or aqueous solution in the second mixing described above] When Vf [unit: mL] is defined as (final volume) (volume after measuring up), the ratio value (Vf / Va) obtained by dividing Vf by Va is an emulsion solution composed of stable and uniform micelle particles. In view of the purpose of manufacturing, it is preferably in the range of 1 to 500.

That is, it is possible to select the values (capacities) of Va and Vf so that the ratio value (Vf / Va) obtained by dividing Vf by Va is in the range of 1 to 500 from the above purpose. preferable.

Similarly, for the above purpose, the ratio value (Vf / Va) obtained by dividing Vf by Va is more preferably in the range of 2 to 200, and particularly in the range of 5 to 100. preferable.

That is, for the above purpose, the values of Va and Vf (capacities) may be selected so that the ratio value (Vf / Va) obtained by dividing Vf by Va is in the range of 2 to 200. More preferably, the values (capacities) of Va and Vf are particularly preferably selected so as to be in the range of 5 to 100.

The above-mentioned [A] is a step of mixing all or part of a mixture prepared by mixing DGGMR and a surfactant with a certain amount of water or an aqueous solution, but is not particularly limited. All or part of the “mixture prepared by mixing DGGMR and surfactant” may be added to a certain amount of “water or aqueous solution” and mixed, or a certain amount of “water or aqueous solution” may be added to “DGGMR”. The mixture may be added to all or part of the “mixture prepared by mixing the surfactant and the surfactant” and mixed, or other embodiments may be employed.

In addition, a certain amount of water or an aqueous solution is further mixed with the mixed solution after mixing the mixture (mixture of DGGMR and a surfactant) and water or an aqueous solution in the step [A] of [B]. Stage ", but there is no particular limitation. For example," a mixture after mixing the mixture (DGGMR and surfactant) in the stage [A] and water or an aqueous solution "with a certain amount of" It may be added to and mixed with "water or aqueous solution", or a certain amount of "water or aqueous solution" may be added to "the mixture (DGGMR and surfactant mixture) in step [A]" and water or an aqueous solution. The mixture may be added to the mixed solution after mixing and mixed, or other embodiments may be employed.

(3) Method of mixing In the step of mixing all or part of a mixture prepared by mixing DGGMR as a lipase activity measurement substrate and a surfactant with water or an aqueous solution, all or part of the mixture is The method of mixing with water or an aqueous solution is not particularly limited as long as the mixture and the water or aqueous solution are mixed.

In this mixing, the lipase activity measurement substrate is mixed with a solution containing an organic solvent such as alcohol, the lipase activity measurement substrate-containing solution is dropped and mixed with the solution, or the lipase activity measurement Complicated or skillful, such as spray injection of the substrate-containing solution into the solution, stirring the lipase activity measurement substrate solution at high speed with a powerful mixer, or applying ultrasonic waves to the lipase activity measurement substrate solution No special treatment or special equipment is required, such as stirring at a general speed using a general mixer, and mixing may be performed by a normal method, thereby making it a substrate for measuring lipase activity. All or part of the DGGMR / surfactant mixture can be mixed with water or an aqueous solution.

(4) Temperature at the time of mixing In the step of mixing all or part of the mixture prepared by mixing DGGMR as a lipase activity measurement substrate and a surfactant with water or an aqueous solution, all or part of the mixture is mixed. The temperature at the time of mixing with water or an aqueous solution is not particularly limited, but performing this step at a temperature below the cloud point of the surfactant used is intended to produce an emulsion solution consisting of stable and uniform micelle particles. Preferred from above.

And the process which mixes all or one part of the mixture prepared by mixing this DGGMR and surfactant with water or aqueous solution is 10 degreeC lower than the cloud point of the surfactant used for the said objective. It is more preferable to carry out below, and it is especially preferable to carry out at 25 degrees C or less.

In the step of mixing all or part of the mixture prepared by mixing DGGMR and surfactant with water or an aqueous solution, all or part of the mixture of DGGMR and surfactant is mixed with water or aqueous solution. However, it is preferable for the purpose of producing an emulsion solution composed of stable and uniform micelle particles that this step is performed at a temperature equal to or higher than the melting points of DGGMR and the surfactant to be used. .

Then, the step of mixing all or part of the mixture prepared by mixing DGGMR and the surfactant with water or an aqueous solution is more preferably performed at 10 ° C. or more for the above purpose, and 15 ° C. It is particularly preferable to perform the above.

(5) Mixing time In the step of mixing all or part of the mixture prepared by mixing DGGMR as a lipase activity measurement substrate and a surfactant with water or an aqueous solution, all or part of the mixture is The time for mixing with water or an aqueous solution is not particularly limited as long as the mixture of DGGMR and surfactant and the water or aqueous solution are mixed homogeneously.

Usually, this mixing is preferably performed for 5 minutes or more for the purpose of producing an emulsified lipase activity measuring substrate solution composed of stable and uniform micelle particles. In general, 5 minutes is sufficient.

Further, the time for mixing the mixture of DGGMR and the surfactant with water or an aqueous solution is not particularly limited, and may be mixed for several hours, for example, but from the viewpoint that time is also cost. Even if you do it carefully, it usually takes less than 10 minutes.

[2] Reagent activity measuring reagent of the present invention General The reagent for measuring lipase activity of the present invention comprises a lipase containing “1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] as a substrate for measuring lipase activity. A reagent for measuring lipase activity comprising a substrate solution for measuring lipase activity characterized in that the substrate solution for measuring activity contains a reducing agent. (The details of the substrate solution for measuring lipase activity are as described in the above section [1] Substrate solution for measuring lipase activity of the present invention.)

And the reagent for measuring lipase activity of the present invention can prevent the degradation of DGGMR due to the mixed azide and suppress the decrease in the absorbance value obtained by the measurement by the above-mentioned constitution.

II. Lipase activity measuring reagent Configuration of Lipase Activity Measuring Reagent, etc. The lipase activity measuring reagent of the present invention may consist only of the lipase activity measuring substrate solution of the present invention, or the lipase activity measuring substrate solution of the present invention and other constituent reagents. Or a reagent kit.

For the following reasons (a) and (b), the lipase activity measuring reagent of the present invention is preferably a reagent kit comprising the lipase activity measuring substrate solution of the present invention and other constituent reagents.
(A) The substrate for measuring lipase activity [DGGMR] in the present invention is stable at pH 4 or a pH around it, whereas lipase has an optimum activity at pH 8 or a pH near it, and each has a suitable pH. The area is different.
(B) When the lipase activity measurement substrate [DGGMR], colipase, and bile acid or a salt thereof as a lipase activator in the present invention coexist in one reagent, the lipase activity measurement substrate [DGGMR] in the present invention. The stability of is not good.

Therefore, the lipase activity measuring reagent of the present invention is preferably a reagent kit comprising a lipase activity measuring substrate solution and other constituent reagents. In this case, the pH of the reagent containing the lipase activity measuring substrate [DGGMR] is pH 4 Alternatively, the pH of at least one of the other constituent reagents combined therewith is preferably pH 8 or higher, and the lipase activity measurement substrate [DGGMR], colipase, and lipase activation It is preferable that bile acid or a salt thereof as an agent does not coexist in one reagent.

The lipase activity measurement reagent of the present invention is preferably a two-reagent reagent kit comprising a lipase activity measurement substrate solution and one other component reagent.

In this case, it is more preferable to use the other constituent reagent as the first reagent and the lipase activity measurement substrate solution as the second reagent.

In this case, the pH of the other constituent reagent is more preferably pH 8 or higher, and the pH of the substrate solution for lipase activity measurement is more preferably pH 4 or the vicinity thereof.

In addition, the substrate solution for measuring lipase activity does not contain both “colipase” and “bile acid or its salt as lipase activator”, but “colipase” and “lipase activator as More preferably, at least one of “bile acid or salt thereof” is contained in the other constituent reagent.

The reagent for measuring lipase activity of the present invention may be one measured by the end point method (end point method), or one measured by the reaction rate method (rate method), and may be appropriately selected. The reaction rate method (rate method) is preferred.

In the lipase activity measuring reagent of the present invention, the substrate for measuring lipase activity [DGGMR] is brought into contact with the sample and reacted to cause 1,2-o-dilauryl-rac- by a hydrolysis reaction catalyzed by the lipase. Glycerol and glutaric acid- (6′-methylresorufin) -ester are produced, but this glutaric acid- (6′-methylresorufin) -ester is unstable and easily hydrolyzed naturally. '-Methylresorufin (λmax: 580 nm) is produced.

Therefore, the increase in 6'-methylresorufin produced may be measured by measuring absorbance at a wavelength of 580 nm or in the vicinity thereof, and the activity value of lipase contained in the sample may be obtained. In this case, a single wavelength method or a dual wavelength method may be used.

Further, in the lipase activity measurement reagent of the present invention, the temperature during the measurement reaction is a range in which the measurement reaction such as 30 ° C. or 37 ° C. proceeds and the reaction components such as enzymes involved in the measurement reaction are not inactivated, denatured or altered by heat The temperature inside may be set.

In the lipase activity measurement reagent of the present invention, the method for starting the measurement reaction is any method such as a method performed by adding a lipase activity measurement substrate [DGGMR] or the like in the present invention, or a method performed by adding a sample. It may be.

Further, in the lipase activity measurement reagent of the present invention, the measurement may be performed by a method, or may be performed using an apparatus such as an automatic analyzer.

In the lipase activity measuring reagent of the present invention, all or part of the constituent reagents may be a liquid reagent.

The substrate solution for measuring lipase activity of the present invention can be sold alone or used for measuring lipase activity in a sample.

Moreover, the substrate solution for measuring lipase activity of the present invention can be sold in combination with the above-mentioned other constituent reagents or other reagents or used for measuring lipase activity in a sample.
Examples of the other constituent reagents or other reagents include, for example, a buffer solution, a sample diluent, a reagent diluent, a reagent containing a substance for performing calibration (calibration), or for performing quality control. Examples include reagents containing substances.

2. Specific examples of the lipase activity measuring reagent of the present invention Specific examples of the lipase activity measuring reagent of the present invention are given below.

(1) Example 1
(A) First Reagent [Aqueous solution containing the following reagent components at the indicated concentrations; pH 8.3 (20 ° C.)]
Sodium deoxycholate [Lipase activator] 2% (w / v)
Calcium chloride [Lipase activator] 5 mM
Colipase (derived from porcine pancreas; Roche Diagnostics Inc. [Japan]) 375K units / L (5mg / L)
Bicine [Buffer] 40 mM

(B) Second reagent (substrate solution for measuring lipase activity of the present invention) [Aqueous solution containing the following reagent components at the respective concentrations; pH 4.0 (20 ° C.)]
1,2-o-Dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] (Roche Diagnostics Inc. [Japan]) [Lipase activity measurement substrate ] 0.3 mM
Side chain type non-reactive polyether-modified type modified silicone oil 0.3% (w / v)
Hydroxyl ammonium chloride [Reducing agent] 0.5 mM
L-tartaric acid [buffer] 40 mM

(2) Example 2
(A) First Reagent [Aqueous solution containing the following reagent components at the indicated concentrations; pH 8.4 (20 ° C.)]
Sodium taurodeoxycholate [lipase activator] 2% (w / v)
Sodium deoxycholate [Lipase activator] 0.2% (w / v)
Calcium chloride [Lipase activator] 5 mM
Colipase (derived from porcine pancreas; Roche Diagnostics Inc. [Japan]) 150K units / L (2mg / L)
Tris (hydroxymethyl) aminomethane [Tris] [Buffer] 40 mM

(B) Second reagent (substrate solution for lipase activity measurement of the present invention) [Aqueous solution containing the following reagent components at the indicated concentrations]
1,2-o-Dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] (Roche Diagnostics Inc. [Japan]) [Lipase activity measurement substrate ] 0.6 mM
Side chain type non-reactive polyether-modified type modified silicone oil 0.3% (w / v)
Hydroxyl ammonium chloride [Reducing agent] 1 mM
Sodium taurodeoxycholate [lipase activator] 2% (w / v)

[3] Method for measuring lipase activity of the present invention General The method for measuring lipase activity according to the present invention is a lipase comprising 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR] as a substrate for measuring lipase activity. In the method for measuring lipase activity using a substrate solution for activity measurement, a reducing agent is contained in the substrate solution for lipase activity measurement. (The details of the substrate solution for lipase activity measurement are as described in the above-mentioned section [1] Substrate solution for lipase activity measurement of the present invention.) The details of the lipase activity measurement reagent are as follows. (As described in the above section [2] Reagent activity measurement reagent of the present invention].)

And the lipase activity measuring method of this invention can prevent the degradation of DGGMR by the mixed azide and suppress the fall of the light absorbency value obtained by a measurement by said structure.

II. Method for measuring lipase activity Lipase activity measurement method

(1). Sample In the present invention, the sample for measuring the activity of lipase may be any sample that may contain lipase, and is not particularly limited as long as it may contain lipase.
Examples of the sample include a sample derived from a human, an animal, or a plant.

The sample derived from a human or animal is not particularly limited, for example, body fluid such as blood, serum, plasma, semen, spinal fluid, saliva, sweat, tears, ascites, or amniotic fluid of human or animal; urine or stool Excrements such as: organs such as pancreas, liver or stomach; tissues such as hair, skin, nails, muscles or nerves; or cells.

The present invention is suitable when a sample derived from a human or an animal is used as a sample, and particularly suitable when a sample derived from a human is used as a sample.

In addition, the present invention is suitable when a body fluid, organ or tissue is used as a sample, more preferably when a body fluid is used as a sample, and more preferable when blood, serum or plasma is used as a sample. This is particularly suitable when plasma is used as a sample.

In the present invention, since the sample is suitable when it is a liquid, if the sample is not a liquid, pretreatment such as extraction or solubilization may be performed according to a known method to obtain a liquid sample. Good.
Further, the sample may be diluted or concentrated as necessary.

(2) Details of measurement When the lipase activity measurement method of the present invention is used to measure lipase activity in a sample, the measurement may be performed by the end point method (end point method), or the reaction rate. Measurement may be performed by a method (rate method) and may be appropriately selected, but a reaction rate method (rate method) is preferred.

In addition, when measuring the lipase activity in a sample by the lipase activity measuring method of the present invention, the measurement is performed by a one-step method performed by one step, or by two or more multi-steps. Measurement may be performed by appropriately selecting a step method.

When the lipase activity measurement reagent used for measuring the lipase activity is composed of the first reagent, the second reagent, and another reagent (one or two or more reagents), that is, when the reagent comprises three or more reagents. The measurement reaction may be performed through the number of steps necessary to perform the measurement using these reagents (two steps or three or more steps as required), and the lipase activity in the sample may be measured. .

In the method for measuring lipase activity of the present invention, 1,2-o-dilauryl-rac- is obtained by a hydrolysis reaction catalyzed by lipase by bringing a sample into contact with a substrate for measuring lipase activity [DGGMR] and reacting it. Glycerol and glutaric acid- (6′-methylresorufin) -ester are produced, but this glutaric acid- (6′-methylresorufin) -ester is unstable and easily hydrolyzed naturally. '-Methylresorufin (λmax: 580 nm) is produced.

Note that calculating the activity value of lipase contained in a sample from the measured absorbance (or transmittance) or the amount of change in absorbance (or transmittance) is the molar extinction coefficient of 6′-methylresorufin. Such as a method of calculating from the absorbance (or transmittance) measured based on the standard, or a method of calculating by comparing with the absorbance (or transmittance) of a standard substance (standard solution, standard serum, etc.) whose lipase activity value is known A method may be selected as appropriate.

Further, it is preferable to calculate the activity value of lipase contained in the sample by subtracting the reagent blind test (reagent blank) from the absorbance (or transmittance) obtained by measuring the sample.

In the lipase activity measurement method of the present invention, the temperature during the measurement reaction is such that the measurement reaction such as 30 ° C. or 37 ° C. proceeds, and the reaction components such as enzymes involved in the measurement reaction are not deactivated, denatured or altered by heat. What is necessary is just to set the temperature within the range.

In the lipase activity measurement method of the present invention, the measurement reaction may be initiated by any method such as a method performed by adding a lipase activity measurement substrate or the like, or a method performed by adding a sample.

Further, in the lipase activity measurement method of the present invention, the measurement may be carried out by a method, or may be carried out using an apparatus such as an automatic analyzer.

2. Specific examples of the method for measuring lipase activity Specific examples of the method for measuring lipase activity of the present invention are given below.

(1) Lipase activity measuring reagent (a) First reagent The above-mentioned [2] II-2 (1) (a) first reagent was used as the first reagent in a specific example of this measuring method.

(B) Second Reagent The second reagent of (2) II (1) (b) of [2] above was used as the second reagent in the specific example of this measurement method.

(2) Sample Human serum was used as a sample.

(3) Measurement (a) 1st stage The sample of said (2) and the 1st reagent of (a) of said (1) are mixed, and a liquid mixture is prepared.

The amounts of the sample and the first reagent to be mixed may be appropriately determined according to the amount of the second reagent, the activity value of the lipase contained in the sample, and other conditions.
In general, for example, the amount of the sample is preferably 0.5 to 100 μL, and the amount of the first reagent is preferably in the range of 20 to 1,000 μL.

Incubation is performed after preparation of this mixture.
The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.

Moreover, the temperature at the time of incubation should just be the temperature above the temperature which the said liquid mixture freezes.
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, components such as enzymes involved in the measurement reaction will be denatured and inactivated, so the temperature during incubation is below the temperature at which the components such as enzymes involved in the measurement reaction are denatured and deactivated. It is necessary to.
The incubation temperature is usually 2 to 70 ° C., preferably 20 to 37 ° C., more preferably 30 to 37 ° C.
In addition, if the component such as an enzyme involved in the measurement reaction is a heat-resistant component such as a heat-resistant enzyme, the temperature may be higher.

By preparing and incubating the mixed solution of the sample and the first reagent, the lipase contained in the sample comes into contact with the reagent components contained in the first reagent, and activation or activation of the lipase by these components is performed. Done.

(B) Second Stage The second reagent (b) of (1) is mixed with the “mixture of sample and first reagent” prepared in the first stage. This is the final reaction solution.

The amount of the second reagent to be mixed may be appropriately determined according to the amount of the sample, the amount of the first reagent, the activity value of the lipase contained in the sample, the specifications of the analyzer to be used, and other conditions.
In general, for example, the amount of the second reagent is preferably in the range of 10 to 1,000 μL.

Incubation is performed after preparation of this final reaction solution.
The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.

Moreover, the temperature at the time of incubation should just be the temperature above the temperature which freezes the said last reaction liquid.
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, components such as enzymes involved in the measurement reaction will be denatured and inactivated, so the temperature during incubation is below the temperature at which the components such as enzymes involved in the measurement reaction are denatured and deactivated. It is necessary to.
The incubation temperature is usually 2 to 70 ° C., preferably 20 to 37 ° C., more preferably 30 to 37 ° C.
In addition, if the component such as an enzyme involved in the measurement reaction is a heat-resistant component such as a heat-resistant enzyme, the temperature may be higher.

By the preparation and incubation of the final reaction solution, the measurement reaction in the second stage is started following the activation and activation of the lipase in the first stage, and the activity measurement reaction of the lipase contained in the sample is started. It is advanced.

That is, the second reagent (substrate solution for measuring lipase activity), which is an emulsified substrate solution composed of stable and uniform micelle particles, comes into contact with the lipase contained in the sample in this second stage, The hydrolysis reaction catalyzed by lipase produces 1,2-o-dilauryl-rac-glycerol and glutaric acid- (6′-methylresorufin) -ester from the lipase activity measurement substrate [DGGMR].
Since this glutaric acid- (6′-methylresorufin) -ester is unstable, it is easily hydrolyzed naturally to produce 6′-methylresorufin (λmax: 580 nm).

Since the generated 6′-methylresorufin has a maximum absorption wavelength (λmax) of 580 nm, the absorbance (or transmittance) of the final reaction solution derived from this 6′-methylresorufin is 580 nm or near it. It is measured by measuring the absorbance (or transmittance) at the wavelength of.

Next, the activity value of the lipase contained in the sample is calculated from the measured absorbance (or transmittance) or the amount of change in absorbance (or transmittance).

This is a method of calculating from the absorbance (or transmittance) measured based on the molar extinction coefficient of 6′-methylresorufin, or a standard substance (standard solution, standard serum, etc.) whose lipase activity value is known. A method such as a method of calculating by comparing with the absorbance (or transmittance) of is suitably selected.

The activity value of the lipase contained in the above sample was calculated by subtracting the reagent blindness (reagent blank) from the absorbance (or transmittance) of the final reaction solution obtained by measuring the sample. (ΔAbs.) Is preferably used.

[4] Method for suppressing influence of azide on measurement of lipase activity of the present invention The method for suppressing the influence of azide on the measurement of lipase activity of the present invention comprises 1,2-o-dilauryl-rac-glycero-3-glutaric acid- A reducing agent is contained in a lipase activity measurement substrate solution containing (6′-methylresorufin) -ester [DGGMR] as a lipase activity measurement substrate. (The details of the substrate solution for lipase activity measurement are as described in the above-mentioned section [1] Substrate solution for lipase activity measurement of the present invention.) The details of the lipase activity measurement reagent are as follows. As described in the above section “[2] Reagent activity measurement reagent of the present invention.” For details of the lipase activity measurement method, refer to the section “[3] Method for measuring lipase activity of the present invention”. As described in.)

And the suppression method of the influence of the azide with respect to the lipase activity measurement of this invention is a method which can prevent deterioration of DGGMR by the azide mixed by the above-mentioned structure, and can suppress the fall of the absorbance value obtained by measurement. is there.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] (Confirmation of effect of suppressing influence of azide of the present invention-1)
The suppression effect of the influence of the azide in this invention was confirmed.

1. Reagent activity measuring reagent of the present invention The first reagent and the second reagent of the lipase activity measuring reagent of the present invention were prepared.

[1] First Reagent The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to pH 8.4 (20 ° C.) to prepare a first reagent for measuring lipase activity. .

Sodium deoxycholate [Lipase activator] 2% (w / v)
Calcium chloride [Lipase activator] 20 mM
Colipase (derived from porcine pancreas, distributor: Roche Diagnostics Inc. [Japan]) 750K units / L (10mg / L)
Bicine [Buffer] 80 mM

[2] Second reagent (substrate solution for measuring lipase activity)
[1] Azide-added second reagent (the present invention)
In order to confirm the influence of azide contamination on the second reagent (lipase activity measurement substrate solution) of the lipase activity measuring reagent of the present invention, an “azide added second reagent (invention)” in which azide was added was prepared. .

(1) 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester [DGGMR], a substrate for measuring lipase activity (Distributor: Roche Diagnostics) 0.045 grams [g] of Su Co., Ltd. [Japan]) was weighed into a beaker (capacity: 10 mL).

(2) Next, 0.6 g [g of KF-355A (distributor: Shin-Etsu Chemical Co., Ltd. [Japan]), which is a non-reactive modified silicone oil (polyether-modified type) of a side chain type. ] Was weighed and added to the beaker of (1) above.

(3) After the addition of (2), the beaker was stirred at 67 ° C., and the lipase activity measurement substrate [DGGMR] in the beaker and the surfactant KF-355A were mixed.

This mixing (stirring) was carried out for 5 minutes to prepare a “lipase activity measurement substrate [DGGMR] and surfactant mixture”.

In this stirring, the beaker is placed on a multi-stirrer (model: M-3, distributor: As One Co., Ltd. [Japan]), and the rotor in the beaker is marked with a scale “3” of the multi-stirrer. This was done by rotating at

(4) Next, “a certain amount (4.0 mL) of 1 mM chloride was added to the“ mixture of lipase activity measurement substrate [DGGMR] and surfactant ”(all) in the beaker of (3) above while stirring. 40 mM L-sodium tartrate buffer [pH 4.0 (20 ° C.)] containing hydroxylammonium (reducing agent) was added from a micropipette at room temperature (25 ° C.).

Then, after the addition, this stirring is continued for 5 minutes at room temperature (25 ° C.), whereby the above-mentioned “mixture prepared by mixing the lipase activity measurement substrate [DGGMR] and the surfactant” (all ) And the above-mentioned “a constant amount (4.0 mL) of 40 mM L-sodium tartrate buffer solution containing 1 mM hydroxylammonium chloride [pH 4.0 (20 ° C.)]”.

(5) Next, 40 mM L-sodium tartrate buffer containing “the mixture (mixture of lipase activity measurement substrate [DGGMR] and surfactant)” and a fixed amount of 1 mM hydroxylammonium chloride [4) above. 40 mM L-sodium tartrate buffer solution [pH 4.0 (20 ° C.)] containing a certain amount of 1 mM hydroxylammonium chloride was further mixed with “the mixture after mixing with pH 4.0 (20 ° C.)”. The final volume was 200 mL.

(6) By the above operation, the “lipase activity measurement substrate solution (substrate for lipase activity measurement: DGGMR, surfactant: KF-355A, reducing agent: hydroxylammonium chloride) which is the substrate solution for lipase activity measurement of the present invention” Was prepared.
This was designated as “second reagent (present invention)” which is the substrate solution for lipase activity measurement of the present invention.

(7)
10 μL of 0.1% (w / v) aqueous sodium azide solution was added to 10 mL of the “second reagent (invention)” of (6), and “azide-added second reagent (invention)” did.

(8) In the “azide-added second reagent (present invention)” prepared in (1) to (7) above, the concentration of the lipase activity measurement substrate [DGGMR] is 0.3 mM, and the surface activity The concentration of the agent (KF-355A) is 0.3% (w / v), the concentration of the reducing agent (hydroxylammonium chloride) is 1 mM, and the concentration of sodium azide is 0.0001% (w / v) ).
In addition, in this “second azide-added second reagent (invention)”, it was confirmed visually that no concentration gradient or strong turbidity was observed, and that the mixture was homogeneously mixed.

[2] Azide-added second reagent (conventional invention)
In order to confirm the influence of azide contamination on the second reagent (substrate solution for lipase activity measurement) of the lipase activity measurement reagent of the conventional invention, a “second reagent (conventional invention)” in which azide was added was prepared.

(4) Next, the “mixture of lipase activity measurement substrate [DGGMR] and surfactant” (all) in the beaker of (3) above (all) was stirred with “a certain amount (4.0 mL) of 40 mM L -Sodium tartrate buffer [pH 4.0 (20 ° C)] "was added from a micropipette at room temperature (25 ° C).

Then, after the addition, this stirring is continued for 5 minutes at room temperature (25 ° C.), whereby the above-mentioned “mixture prepared by mixing the lipase activity measurement substrate [DGGMR] and the surfactant” (all ) And the above-mentioned “constant amount (4.0 mL) of 40 mM L-sodium tartrate buffer [pH 4.0 (20 ° C.)]”.

(5) Next, “the mixture (mixture of lipase activity measurement substrate [DGGMR] and surfactant)” and a fixed amount of 40 mM L-sodium tartrate buffer [pH 4.0 (20 ° C.)] ] Was further mixed with a certain amount of 40 mM L-sodium tartrate buffer [pH 4.0 (20 ° C.)] to a final volume of 200 mL.

(6) According to the above operation, a “lipase activity measurement substrate solution (lipase activity measurement substrate: DGGMR, surfactant: KF-355A)”, which is a conventional lipase activity measurement substrate solution, was prepared.
This was designated as “second reagent (conventional invention)” which is a substrate solution for lipase activity measurement of the conventional invention.

(7)
10 μL of 0.1% (w / v) sodium azide aqueous solution was added to 10 mL of the “second reagent (conventional invention)” of (6), and “azide-added second reagent (conventional invention)” was obtained. did.

(8) In the “second azide-added second reagent (conventional invention)” prepared in (1) to (7) above, the concentration of the lipase activity measurement substrate [DGGMR] is 0.3 mM, and the surface activity The concentration of agent (KF-355A) is 0.3% (w / v) and the concentration of sodium azide is 0.0001% (w / v).
Further, in this “second azide-added second reagent (conventional invention)”, it was visually confirmed that no concentration gradient or strong turbidity was observed, and that the mixture was homogeneously mixed.

[3] Control Second Reagent A “control second reagent” that is a second reagent (substrate solution for lipase activity measurement) of the lipase activity measurement reagent as a control was prepared. In addition, azide was not added to this “control second reagent”.

(6) By the above operation, a “lipase activity measurement substrate solution (lipase activity measurement substrate: DGGMR, surfactant: KF-355A)” was prepared.
This was used as a “control second reagent” which is a substrate solution for lipase activity measurement as a control.

(7) In the “control second reagent” prepared in (1) to (6) above, the concentration of the lipase activity measurement substrate [DGGMR] was 0.3 mM, and the surfactant (KF-355A) The concentration of is 0.3% (w / v).
Further, in this “control second reagent”, no concentration gradient or strong turbidity was observed, and it was visually confirmed that the mixture was homogeneously mixed.

2. Reagent storage “Azide-added second reagent (invention)” prepared in [1] of [2] above and “Azide-added second reagent” prepared in [2] of [2] above (Conventional invention) ”was stored at 5 ° C. in the dark for 7 days.

3. “(1) Standard substance”, “(2) Standard serum”, and “(3) Control serum” below the samples were used as samples.

(1) Standard substance “Common reference standard substance: JSCC regular enzyme JCCLS CRM-001c” (distributor: Japan Clinical Laboratory Standards Association [Japan]) was used as the “standard substance”.

(2) Standard Serum Commercially available standard serum “Cygnus Auto LIP Standard Serum” [manufacturing number: G501] (distributor: Sinotest Inc. [Japan]) was used as “standard serum”.

(3) Control Serum “CYGNUS AUTO LIP CONTROL” [manufacturing number: G501] (distributor: Sinotest Inc. [Japan]), which is a commercially available control serum for quality control, was used as “control serum”.

4). Measurement of lipase activity in sample “First Reagent” and “Control Second Reagent” and “Second Azide Added Reagent (Invention)” stored in 5 above at 5 ° C. (dark place) for 7 days and “ Using the azide-added second reagent (conventional invention), each sample of the above 3 was measured with a 7180 type general-purpose automatic analyzer (distributor: Hitachi High-Technologies Corporation [Japan]).

(1) In the 7180 general-purpose automatic analyzer, for each of the three samples (“(1) standard substance”, “(2) standard serum”, and “(3) control serum”), these samples In addition, 160 μL of the “first reagent” prepared in [1] of 1 above was added as a first reagent, and reacted at 37 ° C.

(2) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), the “azide addition second reagent ( 96 μL of the “invention” ”was added as a second reagent and reacted at 37 ° C.

(3) Next, the change in absorbance from 20 points (340.510 seconds after addition of the first reagent) to 24 points (411.887 seconds after addition of the first reagent) is measured at the main wavelength of 570 nm and the sub wavelength of 700 nm. It was measured. (Measurement of change in absorbance based on increased concentration of 6'-methylresorufin produced according to the activity value of lipase contained in the sample)

(4) Moreover, the physiological saline was used for the measurement of a reagent blind test (reagent blank).
Except for using this physiological saline as a sample, the procedure described in the above (1) to (3) was performed, and the amount of change in absorbance when the physiological saline was measured was measured. (Measurement of absorbance change in reagent blind test)

(5) Next, subtract the absorbance change amount of the reagent between the points obtained in (4) from the absorbance change amount between the points for each of the three samples obtained in (3). Then, “the value of the difference in absorbance change of the sample” was determined.

(6) In addition, except that the second reagent in (2) is changed from “second azide-added second reagent (present invention)” to “azide-added second reagent (conventional invention)” stored in 2 above, The operation was carried out as described in the above (1) to (5), and the lipase activity value (value of difference in absorbance change of the sample) of each of the three samples was determined.

(7) Also, the second reagent in (2) above is changed from “second azide added second reagent (present invention)” to “control second reagent” prepared in [1] [2] [3]. Were operated as described in (1) to (5) above, and the lipase activity value (value of difference in absorbance change of the sample) of each sample in 3 was obtained.

5). Measurement Results Table 1 shows the lipase activity values (difference in the amount of change in absorbance of the samples) of the three samples measured and determined in 4 above.

The unit of the measured value (lipase activity value of each sample) shown in Table 1 is “absorbance difference (ΔAbs.)”.

In Table 1, the values in parentheses are the measured values (lipase activity value [value of difference in absorbance change of sample]), and azide is not added as the second reagent. The value divided by the measured value (lipase activity value [value of difference in absorbance change of sample]) of each sample when “2 Reagents” is used is expressed in percent.

6). Discussion (1) From Table 1, the measured value (lipase activity [value of difference in absorbance change of sample]) when using “second azide-added second reagent (conventional invention)” as the second reagent is Compared to the case of using “control second reagent” with no azide added as two reagents, “(1) standard substance” is 67%, and “(2) standard serum” is 67%. And in "(3) control serum", it is 70%, and it can be seen that the absorbance obtained by the measurement is greatly reduced.

(2) On the other hand, from Table 1 as well, the measured value (lipase activity value [value of difference in absorbance change of sample) when “second azide-added second reagent (present invention)” is used as the second reagent. ]) Is 105% in “(1) Standard substance” and “(2) Standard serum”, compared with the case where “control second reagent” not added with azide is used as the second reagent. Is 101%, and it is 101% in “(3) Control serum”, which shows that there is almost no difference.

(3) That is, in the present invention, it was confirmed that the degradation of DGGMR due to the mixed azide can be prevented and the decrease in the absorbance value obtained by the measurement can be suppressed.

[Example 2] (Confirmation of the effect of suppressing the influence of the azide of the present invention-2)
The suppression effect of the influence of the azide in this invention was confirmed again.

In addition, this 1st reagent was prepared 3 times in total separately, and these were made into the 1st lot, the 2nd lot, and the 3rd lot of the 1st reagent of a lipase activity measuring reagent, respectively.

[2] Second reagent (substrate solution for measuring lipase activity)
[1] Azide-added second reagent (the present invention)
In order to confirm the influence of azide contamination on the second reagent (lipase activity measurement substrate solution) of the lipase activity measurement reagent of the present invention, a total of 3 “azide addition second reagent (invention)” added with azide was added. A lot was prepared.

(4) Next, “a certain amount (4.0 mL) of 0.6 mM” was added to the “mixture of lipase activity measurement substrate [DGGMR] and surfactant” (all) in the beaker of (3) above while stirring. Of 40 mM L-sodium tartrate buffer [pH 4.0 (20 ° C.)] containing hydroxylammonium chloride (reducing agent) was added from a micropipette at room temperature (25 ° C.).

Then, after the addition, this stirring is continued for 5 minutes at room temperature (25 ° C.), whereby the above-mentioned “mixture prepared by mixing the lipase activity measurement substrate [DGGMR] and the surfactant” (all ) And the above-mentioned “a fixed amount (4.0 mL) of 40 mM L-sodium tartrate buffer solution containing 0.6 mM hydroxylammonium chloride [pH 4.0 (20 ° C.)]”.

(5) Next, 40 mM L-sodium tartrate buffer containing “the mixture (mixture of lipase activity measurement substrate [DGGMR] and surfactant)” and a fixed amount of 0.6 mM hydroxylammonium chloride in the above (4). In addition, a 40 mM L-sodium tartrate buffer solution [pH 4.0 (20 ° C.)] containing a certain amount of 0.6 mM hydroxylammonium chloride was added to the solution [mixture after mixing with the solution [pH 4.0 (20 ° C.)]. To a final volume of 200 mL.

(6) By the above operation, the “lipase activity measurement substrate solution (substrate for lipase activity measurement: DGGMR, surfactant: KF-355A, reducing agent: hydroxylammonium chloride) which is the substrate solution for lipase activity measurement of the present invention” Was prepared.
This was designated as the first lot of the “second reagent (present invention)” which is the lipase activity measurement substrate solution of the present invention.

(7)
10 μL of 0.1% (w / v) aqueous sodium azide solution was added to 10 mL of the “second reagent (invention)” in the above (6), and the “azide-added second reagent (invention)” was added. The first lot was used.

(8) Further, the substrate solution for measuring lipase activity of the present invention, which is the “lipase activity measuring substrate solution (lipase activity measuring)”, is further performed twice as described in the above (1) to (7). Substrate for use: DGGMR, surfactant: KF-355A, reducing agent: hydroxylammonium chloride) "and sodium azide added separately.
These were designated as the second lot and the third lot of the “second azide-added second reagent (invention)”, respectively.

(9) Lipase activity measurement in each of the first lot, the second lot, and the third lot of the “second azide-added second reagent (present invention)” prepared in the above (1) to (8) The substrate [DGGMR] concentration is 0.3 mM, the surfactant (KF-355A) concentration is 0.3% (w / v), and the reducing agent (hydroxylammonium chloride) concentration is 0.6 mM. And the concentration of sodium azide is 0.0001% (w / v).
Further, in any of these “second azide-added second reagents (the present invention)”, no concentration gradient or strong turbidity was observed, and it was visually confirmed that they were uniformly mixed.

[2] Azide-added second reagent (conventional invention)
In order to confirm the influence of azide contamination on the second reagent (lipase activity measurement substrate solution) of the lipase activity measurement reagent of the conventional invention, a total of 3 lots of “second reagent (conventional invention)” with azide added were prepared. .

(6) According to the above operation, a “lipase activity measurement substrate solution (lipase activity measurement substrate: DGGMR, surfactant: KF-355A)”, which is a conventional lipase activity measurement substrate solution, was prepared.
This was designated as the first lot of the “second reagent (conventional invention)” which is a substrate solution for lipase activity measurement of the conventional invention.

(7)
10 μL of 0.1% (w / v) aqueous sodium azide solution was added to 10 mL of the “second reagent (conventional invention)” of (6), and the “azide-added second reagent (conventional invention)” was added. The first lot was used.

(8) Further, the substrate solution for measuring lipase activity according to the conventional invention, ie, “a substrate solution for measuring lipase activity (measurement of lipase activity)” is further performed twice as described in the above (1) to (7). Substrate for use: DGGMR, surfactant: KF-355A) ”with sodium azide added separately.
These were designated as the second lot and the third lot of the “second azide-added second reagent (conventional invention)”, respectively.

(9) In each of the first lot, the second lot, and the third lot of the “azide-added second reagent (conventional invention)” prepared in the above (1) to (8), the lipase activity was measured. The concentration of the substrate for use [DGGMR] is 0.3 mM, the concentration of the surfactant (KF-355A) is 0.3% (w / v), and the concentration of sodium azide is 0.0001% (w / V).
Further, in any of these “second azide-added second reagents (conventional invention)”, no concentration gradient or strong turbidity was observed, and it was visually confirmed that they were uniformly mixed.

[3] Control second reagent (the present invention)
A total of 3 lots of “control second reagent (invention)” which is the second reagent (substrate solution for measuring lipase activity) of the lipase activity measurement reagent as a control were prepared. In addition, azide is not added to this “control second reagent (invention)”.

(4) Next, “a certain amount (4.0 mL) of 0.6 mM” was added to the “mixture of lipase activity measurement substrate [DGGMR] and surfactant” (all) in the beaker of (3) above while stirring. 40 mM L-sodium tartrate buffer solution [pH 4.0 (20 ° C.)] containing hydroxylammonium chloride was added from a micropipette at room temperature (25 ° C.).

(6) By the above operation, a “lipase activity measurement substrate solution (lipase activity measurement substrate: DGGMR, surfactant: KF-355A)” was prepared.
This was designated as the first lot of “control second reagent (invention)” which is a lipase activity measurement substrate solution as a control.

(7) Further, by performing the operation twice more as described in the above (1) to (6), a “lipase activity measurement substrate solution (lipase activity measurement) which is a lipase activity measurement substrate solution as a control. Substrate for use: DGGMR, surfactant: KF-355A) ”was prepared twice separately.
These were designated as the second lot and the third lot of the “control second reagent (invention)”, respectively.

(8) The lipase activity measurement substrate in each of the first lot, the second lot, and the third lot of the “control second reagent (present invention)” prepared in the above (1) to (7) The concentration of [DGGMR] is 0.3 mM, the concentration of surfactant (KF-355A) is 0.3% (w / v), and the concentration of reducing agent (hydroxylammonium chloride) is 0.6 mM. It is.
Further, in any of these “control second reagents (present invention)”, no concentration gradient or strong turbidity was observed, and it was visually confirmed that they were uniformly mixed.

[4] Control second reagent (conventional invention)
A total of 3 lots of “control second reagent (conventional invention)” which is the second reagent (substrate solution for lipase activity measurement) of the lipase activity measurement reagent as a control were prepared. In addition, azide is not added to this “control second reagent (conventional invention)”.

(6) By the above operation, a “lipase activity measurement substrate solution (lipase activity measurement substrate: DGGMR, surfactant: KF-355A)” was prepared.
This was designated as the first lot of “control second reagent (conventional invention)” which is a substrate solution for measuring lipase activity as a control.

(7) Further, by performing the operation twice more as described in the above (1) to (6), a “lipase activity measurement substrate solution (lipase activity measurement) which is a lipase activity measurement substrate solution as a control. Substrate for use: DGGMR, surfactant: KF-355A) ”was prepared twice separately.
These were designated as the second lot and the third lot of the “control second reagent (conventional invention)”, respectively.

(8) The substrate for measuring lipase activity in each of the first lot, the second lot and the third lot of the “control second reagent (conventional invention)” prepared in the above (1) to (7) The concentration of [DGGMR] is 0.3 mM, and the concentration of surfactant (KF-355A) is 0.3% (w / v).
Further, in any of these “control second reagents (conventional invention)”, no concentration gradient or strong turbidity was observed, and it was visually confirmed that they were uniformly mixed.

2. Reagent storage The first lot, the second lot and the third lot of the “second azide-added second reagent (invention)” prepared in [1] of [1] in [1] above, and the [2] in [1] above. The first lot, the second lot, and the third lot of the “second azide-added second reagent (conventional invention)” prepared in 2) were stored at 5 ° C. in the dark for 7 days.

3. “(1) Standard substance”, “(2) Controlled serum-1”, “(3) Controlled serum-2”, “(4) Controlled serum-3”, “(5) Controlled serum-4” below the sample , “(6) Standard serum”, “(7) Control serum-5”, and “(8) Pooled serum” were used as samples, respectively.

(2) Control serum-1
“Liquid control serum I Wako C & C” [manufacturing number: 1515I] (distributor: Wako Pure Chemical Industries, Ltd. [Japan]), which is a commercially available control serum for quality control, was used as “control serum-1”.

(3) Control serum-2
“Liquid control serum II Wako C & C” [manufacturing number: 1515II] (distributor: Wako Pure Chemical Industries, Ltd. [Japan]), which is a commercially available control serum for quality control, was used as “control serum-2”.

(4) Control serum-3
“Aalto Control IR” (distributor: Sinotest Inc. [Japan]), which is a commercially available control serum for quality control, was used as “control serum-3”.

(5) Control serum-4
“Aalto Control II S” (distributor: Sinotest Co., Ltd. [Japan]), which is a commercially available control serum for quality control, was used as “control serum-4”.

(6) Standard Serum Commercially available standard serum “Cygnus Auto LIP Standard Serum” [manufacturing number: G501] (distributor: Sinotest Inc. [Japan]) was used as “standard serum”.

(7) Control serum-5
“CYGNUS AUTO LIP CONTROL” [manufacturing number: G501] (distributor: Sinotest [Japan]), which is a commercially available control serum for quality control, was used as “control serum-5”.

(8) A collection of pooled serum human serum was used as “pooled serum”.

4). Measurement of lipase activity in a sample “First Reagent”, “Control Second Reagent (Invention)” and “Control Second Reagent (Conventional Invention)” and the above 2 stored at 5 ° C. (dark place) for 7 days 7180 using the azide-added second reagent (invention) (first lot to third lot) and the azide-added second reagent (conventional invention) (first lot to third lot) Each of the three samples was measured with a general-purpose automatic analyzer (distributor: Hitachi High-Technologies Corporation [Japan]).

(1) In the above-mentioned 7180 general-purpose automatic analyzer, for each of the three samples, 2.6 μL of these samples is added to the first lot of the “first reagent” prepared in [1] of 1 above. 160 μL was added as a first reagent and allowed to react at 37 ° C.

(6) In addition, except that the second reagent in the above (2) is changed from the first lot to the second lot of the “second azide-added second reagent (present invention)” stored in the above 2, the above (1) to The operation was performed as described in (5), and the lipase activity value (the value of the difference in absorbance change of the sample) of each of the three samples when the second lot was used as the second reagent was determined.

(7) In addition, except that the second reagent in (2) is changed from the first lot to the third lot of the “second azide-added second reagent (invention)” stored in 2 above, the above (1) to The operation was performed as described in (5), and the lipase activity value (value of the difference in absorbance change of the sample) of each of the three samples when the third lot was used as the second reagent was determined.

(8) In addition, except that the second reagent in (2) is changed from “second azide-added second reagent (present invention)” to “azide-added second reagent (conventional invention)” stored in 2 above, Operate as described in (1) to (7) above, and use the first lot to the third lot as the second reagent. The lipase activity value of each of the three samples (the difference in the change in absorbance of the sample) Value).

(9) In addition, the second reagent in (2) above was prepared as “control second reagent (invention)” prepared in [3] of [1] in [2] above from “azide-added second reagent (invention)”. The lipase activity value of each sample (sample 3) when the first lot to the third lot were used as the second reagent by performing the operations as described in the above (1) to (7) except that Value of difference in absorbance change).

(10) In addition, the second reagent in (2) above was prepared as “control second reagent (conventional invention)” prepared in [1] [2] [4] from “azide added second reagent (present invention)”. The lipase activity value of each sample (sample 3) when the first lot to the third lot were used as the second reagent by performing the operations as described in the above (1) to (7) except that Value of difference in absorbance change).

5). Measurement Results Table 2 shows the lipase activity values (values of the difference in absorbance change of the samples) of the three samples measured and determined in 4 above.

The unit of the measured value (lipase activity value of each sample) shown in Table 2 is “absorbance difference (ΔAbs.)”.

In Table 2, the values in parentheses are measured values (lipase activity value [difference in change in absorbance of sample] when “second azide added reagent (invention)”) is used as the second reagent. Value)) is the measured value (lipase activity [value of difference in absorbance change of sample]) when using “control second reagent (invention)” to which no azide is added as the second reagent. The value obtained by dividing the value by percentage, or the measured value when the “second reagent added with azide (conventional invention)” is used as the second reagent (lipase activity value [value of difference in absorbance change of sample]) Divided by the measured value (lipase activity value [value of difference in absorbance change of the sample]) when “control second reagent (conventional invention)” to which azide is not added as the second reagent is used. It is expressed as a percentage.

6). Discussion (1) From Table 2, the measured value (lipase activity value [value of difference in absorbance change of sample]) when “second azide added reagent (conventional invention)” is used as the second reagent is Compared with the use of “control second reagent (conventional invention)” with no azide added as two reagents, 67% to 76% in the first lot and 65% to 76% in the second lot In the third lot, it is 65% to 79%, and it can be seen that the absorbance obtained by the measurement is greatly reduced.

(2) On the other hand, from Table 2, the measured value (lipase activity value [value of the difference in absorbance change of the sample) when “second azide added second reagent (present invention)” is used as the second reagent. ]) Is 96% to 100% in the first lot, compared with the case where the “control second reagent (invention)” to which no azide is added as the second reagent, and in the second lot, It is 96% to 99%, and it is 96% to 100% in the third lot, and it can be seen that there is almost no difference.

(3) That is, in the present invention, it was confirmed again that the degradation of DGGMR due to the mixed azide can be prevented and the decrease in the absorbance value obtained by the measurement can be suppressed.

Claims

A lipase activity measurement substrate solution containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate contains a reducing agent. A substrate solution for measuring lipase activity.
A lipase activity measuring reagent comprising the lipase activity measuring substrate solution according to claim 1.
In a lipase activity measurement method using a lipase activity measurement substrate solution comprising 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate, A method for measuring lipase activity, comprising adding a reducing agent to a substrate solution for measuring lipase activity.
A lipase activity measurement substrate solution containing 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester as a lipase activity measurement substrate contains a reducing agent. A method for suppressing the influence of azide on lipase activity measurement.