JPH04326046A - Method for measuring living organism constituent and reagent kit for measurement - Google Patents

Abstract

PURPOSE:To enable liberation of a substance which is not soluble to water easily or an unstable substance into an aqueous solution to be accelerated by allowing a non-ion interface activator to coexist in a measurement system. CONSTITUTION:A method for measuring a living organism constituent and a reagent kit for measuring the living organism constituent accelerate, as a dissolution co-agent of a substance which is not soluble to water easily or as a stabilizer of an unstable substance, liberation of a substance which is not soluble to water easily or an unstable substance (guest) from cyclodextrin when measuring the living organism constituent by using a reagent where the cyclodextrin is used. That is, nonionic surfactants such as bridge 35 and triton X are allowed to coexist in the measurement system.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring biological components and a reagent kit for measuring biological components. More specifically, biological components are measured using reagents in which cyclodextrins (hereinafter also referred to as CD) are used as solubilizers for substances that are poorly soluble in water or as stabilizers for unstable substances. A biocomponent measuring method and biocomponent devised to promote the release of poorly water-soluble substances or unstable substances into an aqueous solution by coexisting a nonionic surfactant in the measurement system. This invention relates to a measurement reagent kit.

0002

[Prior Art and Problems to be Solved by the Invention] Generally, water-based biological component measuring reagents used for biochemical measurements such as enzyme reactions include enzyme reaction substrates, coenzymes, inhibitors, coloring substances such as dyes, etc. It is necessary to uniformly dissolve (contain) substances that are poorly soluble or unstable in water, such as preservatives. Various attempts have been made to dissolve poorly soluble or unstable substances in water at high concentrations, but there is no perfect method.
For this reason, there are still some substances that cannot be used as reagents for measuring biological components or are not widely used. In addition, these poorly water-soluble substances are usually solubilized using clever methods when preparing reagents for measuring biological components, but they tend to crystallize, precipitate, hydrolyze, oxidize, etc. over time. Once crystallization or precipitation occurs, the concentration of the substance decreases, and the substance loses its function as a highly accurate reagent for measuring biological components, thereby losing its value.

[0003] As one of the effective methods for dissolving such poorly soluble substances in biological component measuring reagents and preventing crystallization or precipitation over time, it has been conventionally known that substances that are poorly soluble in water have been used. A method of coexisting a substance and CD (or CD derivative) or containing them as an clathrate compound has been published (Japanese Patent Laid-Open No. 57-74099 is a Japanese Patent Application Laid-Open No. 60-16).
No. 0896, JP-A-61-260900, JP-A-63
-129999, Japanese Patent Application No. 63-144717).

[0004] In some of these methods, the solubility of the CD itself is too low to actually dissolve poorly soluble substances at high concentrations, or the CD derivatives are extremely expensive and are not practical. Although some of them were completely unusable, they had the following common drawbacks. (1) Substances introduced into the measurement system with improved solubility using CDs and CD derivatives are clathrated by CDs, so they are difficult to become free and must react (act). It does not show sufficient activity as expected under certain conditions. In other words, even if the poorly soluble or unstable substance is contained in the measurement system at a sufficient concentration for calculation due to the solubilization effect of CD, the effective concentration actually decreases due to the inclusion effect of CD. There will be a shortage. (2) It has been known for some time that when CDs are used to increase the solubility of a dye substance, the color tone may change (the absorption spectrum may shift). This is often caused by the fact that the dye introduced into the measurement system is clathrated by the CD and is therefore difficult to become free. This change in color tone is often unfavorable in terms of measurement operations when using clinical test reagents or general analytical reagents, and has been an issue to be solved in reagents using CDs.

[0005] Now, the inventor previously
In Publication No. 97, a method for measuring biological components and a reagent kit for measuring biological components were published, in which a fatty acid was present in the measurement system to promote the release of the poorly soluble or unstable substance (guest) from the CD. There is a fact. When fatty acids are used in the reaction system under alkaline conditions, the molecular weight is relatively small, so it is possible to prepare a reagent with a sufficient molar concentration and the desired effect can be fully exerted. In this system, the solubility of the fatty acid itself is significantly reduced, so there is room for improvement as a substance that promotes guest release.

[0006] The inventor further searched intensively for a substance that promotes guest release, and as a result found that a nonionic surfactant is a substance suitable for this purpose, and completed the present invention. That is, it has been found that if a nonionic surfactant is present in the final reaction system, it can be used as an effective substance for promoting guest release in reaction systems (reagents) in a wide range of pH ranges, whether acidic or alkaline.

[0007]

[Means for solving the problem] The present invention provides the following (1)
The present invention is constituted by claims (6) to (6). (1) When measuring biological components using a biological component measuring reagent containing cyclodextrins as a solubilizer for substances that are poorly soluble in water or as a stabilizer for unstable substances, non-ionic substances must be added to the measurement system. A biological component measuring method characterized by coexisting a surfactant. (2) The living organism described in (1) above, wherein α-cyclodextrin or a derivative of α-cyclodextrin is used as the cyclodextrin, and a surfactant having a linear alkyl in its hydrophobic group is used. Component measurement method. (3) The organism described in (1) above, wherein β-cyclodextrin or a derivative of β-cyclodextrin is used as the cyclodextrin, and a surfactant having an alkylphenyl group in its hydrophobic group is used. Component measurement method. (4) A reagent kit for measuring biological components comprising at least the following two groups of reagents (A) and (B). (A) A reagent containing cyclodextrins as a solubilizing agent for substances poorly soluble in water or as a stabilizer for unstable substances (B) A reagent containing a surfactant (5) As a cyclodextrin, using α-cyclodextrin or α-cyclodextrin derivatives,
The reagent kit for biological component measurement described in (4) above, wherein a surfactant having a linear alkyl group in its hydrophobic group portion is used. (6) using β-cyclodextrin or β-cyclodextrin derivative as the cyclodextrin,
The reagent kit for measuring biological components as described in (4) above, wherein a surfactant having an alkylphenyl group in its hydrophobic group is used.

[0008] In the present invention, a substance poorly soluble in water,
Alternatively, when an unstable substance is stably present as an aqueous solution at a high concentration, CD or a CD derivative is used to give the desired reagent concentration, and nonionic surfactants are used in the final measurement system or reaction system. Let the agents coexist,
A substitution reaction is performed between the guest molecule that has already formed an inclusion complex with CD and the nonionic surfactant molecule, and the guest forms a clathrate with CD.
In addition to eliminating the negative effects caused by similar products, such as color changes, the activity concentration of the guest (original active substance) is increased, and a more effective measurement system and reaction system are realized by combining reagents. It is.

[0009] In the present invention, a poorly water-soluble substance or an unstable substance refers to a relatively low-molecular organic compound described in the following (a) to (f). The present invention is particularly suitable for use with poorly soluble and unstable enzyme substrates such as tyrosine, tryptophan, adenosine, xanthine, and uric acid, and is further characterized in that it detects nitroaniline and nitrophenol derivatives. The configuration of the present invention is also extremely suitable for reaction systems in which (B) Substrates for enzyme reactions (A) Slightly soluble amino acids such as phenylalanine, tyrosine, tryptophan, etc. (B) Slightly water-soluble nucleobases such as adenine, guanine, thymine, cytosyl, uracil, hypoxanthine, xanthine, uric acid, inosine, etc. or its metabolites (C) Low-molecular-weight poorly soluble hormones such as thyroxine, adrenaline, noradrenaline, catecholamines, and melatonin (D) Steroid hormones such as testosterone, aldosterone, corticosterone, progesterone, and estriol (B) Coenzymes (vitamins) ): Nucleotide coenzymes such as nicotinamide adenine dinucleotide and flavin adenine dinucleotide, non-nucleotides such as pyridoxal phosphate, biotin, folic acid, lipoic acid, retinal, carotene, α-tocopherol, vitamin K3, ubiquinone, and vitamin D. System coenzyme (c) Pigments: (A) Phenol, hydroquinone, catechol, pyrogallol, vanillin, etc. and their derivatives, (B) Acid anilide derivatives of nitroaniline, chloronitroaniline, nitrophenol, ester-bonded derivatives of chloronitrophenol, or Ether bond derivative (C
) Phenolphthalein, acridine, phenazine, fluorescein, rhodamine, naphthalene, pyrene, anthracene, umbelliferone, coumarin derivatives and hapten (d) inhibitors labeled with them: dicoumarol, warfarin, thiouracil, 2,4-dinitrophenol , colchicine, p-
Enzymes or metabolic inhibitors such as mercuric chlorobenzoate (e) Preservatives, antibiotics: thymol, chloramphenicol (f) Others: (A) Chelating agents such as o-phenanthroline, 2,2-dipyridyl, bathocuproine, etc. (B) Lipids such as triglycerides and cholesterol

0
[010] In the present invention, CDs include α-, β
-, unmodified CD such as γ-CD, maltosylated α-, β-
So-called branched C such as CD, glucosylated α-, β-CD, etc.
Refers to CD derivatives into which side chains have been introduced to improve water solubility, such as D derivatives and hydroxypropyl-CD.

[0011] The surfactant used in the present invention basically has a hydrophilic group composed of a nonionic polyoxyethylene, a sugar alcohol such as sorbitol, or a derivative thereof,
The hydrophobic group consists of an alkyl group with a prime number of 4 or more, or an alkylphenyl group, and both residues are covalently bonded with an ether bond or an ester bond (both residues may be bonded with a phosphate ester bond via phosphoric acid). Refers to something that has a structure. When α-CD or its derivatives are used as CDs, a nonionic surfactant whose hydrophobic group is a straight-chain alkyl group (e.g. Briji series) is added to CDs with β-CD.
When D or a derivative thereof is used, the hydrophobic group portion is an alkyl group and preferably an alkylphenyl group (e.g. Tr
iton-X series), alone or in combination. Furthermore, a plurality of nonionic surfactants may be used. Furthermore, the concentration of the nonionic surfactant to be allowed to coexist, especially the molecular concentration of the hydrophobic group moiety, is sufficient to exhibit the effect, but guests whose nonionic surfactant molecules are included in CDs Because of the need to replace molecules, it is practical to make it at least higher than the guest molar concentration. Further added C
It is often more preferable to add the hydrophobic group portion of the nonionic surfactant to an extent exceeding the molar concentration of Group D, since most of the guest molecules can be liberated into the reaction system. It is desirable that the concentration of the nonionic surfactant added is at least greater than the concentration of CDs or the concentration of guest molecules, whichever has a smaller molar concentration.

[0012] The present invention is applied to γ as a biochemical analysis item.
-glutamyltransferase (EC2.3.2.2
, hereinafter referred to as GT) activity measurement system will be explained as follows. It is known that the Km (Michaelis-Menten constant) of the enzymatic activity γ-glutamyltransferase (γ-GTP), which uses γ-L-glutamyl-p-nitroanilide (γ-GpNA) as a substrate, is approximately 1 mM. It is being From this value of Km, γ-GTP
In order to accurately measure activity, the concentration of the substrate contained in the reagent needs to be several mM or more. However, pH 4~
At pHs other than 5, the solubility can be relatively increased, but the rate of hydrolysis becomes even greater, so it cannot be stored as a reagent at pHs other than the above. pH4
~ Near neutrality, the solubility of γ-GpNA is minimal, 2 m
In order to prepare a practically usable reagent and store it at a low temperature because it dissolves at less than M (4°C), CD (in this case, the solubility of CD itself is improved, for example, maltosylated CD) is added to increase the solubility. needs to be made larger. However, when using the substrate solution to which CD has been added, there are the following drawbacks. ■ Enzyme activity is lower than expected. This is due to the low affinity between the enzyme (GT) and the substrate, which is a problem with the properties of the enzyme whose activity you want to measure, so the substrate cannot be taken away from the CD inclusion, and the calculation is insufficient. This is due to the phenomenon that although the substrate is supplied at a high concentration, the enzyme reaction system is not provided with a substantially high concentration of the substrate. This phenomenon was exposed as a contradiction in a practical system where an enzyme with a large Michaelis constant and its substrate were dissolved in a highly concentrated aqueous solution by CD because the substrate was poorly soluble. (2) In particular, when the CD composition contains α-CD or a derivative thereof, the absorbance of the reagent blank becomes significantly high. This is because a dye molecule having a nitro group and a benzene ring, or its derivative, is included in CD, causing a spectral shift in the red direction, which improves the sensitivity of the measurement. This is due to the phenomenon of increasing the absorbance of a reagent blank in a reaction system containing a large amount of substrate, ie, a dye derivative. This is particularly noticeable when a CD containing α-CD or a derivative thereof is added to the reagent. Therefore, by allowing a nonionic surfactant to coexist in the reaction system and easily releasing γ-GpNA from CDs, the above-mentioned problems can be solved at once, and extremely accurate measurements can be performed.

[0013]

[Operation] In the present invention, when a poorly soluble or unstable substance in water is stably present as an aqueous solution at a high concentration, CD or a CD derivative is used to provide the desired reagent composition. In the final measurement system, a non-ionic surfactant is coexisting and a substitution reaction is carried out with the guest, which eliminates the adverse effects of CD, such as color change, and improves the effectiveness of the guest. It increases the concentration.

[0014]

[Example 1] <Effects of surfactants on CD derivatives> *In order to confirm the effects of various surfactants on CD derivatives, the following reagents 1 and 2 were prepared, and the following operations were performed to inhibit γ-GTP. The activity value was determined. Reagent 1: 100mM containing 80mM glycylglycine
A uniform 25mM of the nonionic surfactant described in the following ■ to ■ was added to the Tris-HCl buffer solution to adjust the pH to 8.3, and a solution containing no surfactant (no additive,
Control), a total of 9 types (hereinafter referred to as R1-〓, R1-■, etc.)
. ■Sodium caprylate ■Bridge 35 (polyoxyethylene-n-alkyl ether) ■Tween 20 (polyoxyethylene sorbitan monolaurate) ■Polyoxyethylene (n=15) nonylphenyl
Ether■Polyoxyethylene (n=20)・nonylphenyl・
Ether ■Triton X100 (Triton-X100: polyoxyethylene (n≒10)-pt-octylphenol) ■Triton
: Polyoxyethylene (n≒40)-pt-octylphenol) Reagent 2: ■80mM glucosyl α-C
D (G-α-CD), a substrate solution containing 20 mM γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride (hereinafter referred to as R2-■), ■ 80 mM glucosyl-β-CD ( G-β-CD) and 20mM γ-L-
A substrate solution containing glutamyl-p-nitroanilide (γ-GpNA) hydrochloride (hereinafter referred to as R2-■). *Procedure: 25 μL of commercially controlled serum (γ-GTP activity value according to standard method is 47 IU/L) and R1-〓～R1
1000 μL of each of R2-■ and R2-■ are mixed together and heated at 37° C. for 5 minutes, and then 250 μL of each of R2-■ and R2-■ are added to start the reaction. Wavelength 405nm after reaction start
The γ-GTP activity value in the serum due to each reagent is determined from the molecular extinction coefficient of the reaction product P-nitroanilide determined in advance.

*The measurement results are shown in Table 1 (at the end of the volume). In addition,
The percentage in the table represents the ratio to the standard method (activity value of this method/
47 IU). Moreover, the value in parentheses in the activity value column represents the ratio when the activity value without additives is set as 100.

According to the results in Table 1, when the α-form is used as CD, it is particularly preferable to use a nonionic surfactant having a linear alkyl in the hydrophobic group; It can be seen that when the β-form is used as the β-form, those having an alkylphenyl group in the hydrophobic group are particularly excellent. The above results were similar to those measured using maltosyl-α-CD and maltosyl-β-CD.

[0017]

[Example 2] <Study of amount of surfactant added> *The following reagent (R) was prepared. R1-A: 100m containing 80mM glycylglycine
Bridge 35 (linear alkyl type) was added to M Tris-HCl buffer (pH 8.3) at 0,5,10,15,20,2
Added at concentrations of 5, 30 and 50mM. R1-B: 100m containing 80mM glycylglycine
Triton X-10 in M Tris-HCl buffer (pH 8.3)
0 (alkylphenyl group type), 0,5,10,1
Added at concentrations of 5, 20, 25, 30 and 50mM. R2-A: 80mM glucosyl-α-CD (G-α-
CD) and a substrate solution containing 20 mM γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride. R2-B: 80mM glucosyl-β-CD (G-β-
CD) and a substrate solution containing 20 mM γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride. *Procedure: 25 μL of the commercially available control serum used in Example 1 (with a γ-GTP activity value of 47 IU/L according to the standard method) and R
1000 μL of 1-A was mixed, and after heating at 37° C. for 5 minutes, 250 μL of R2-A was added to start the reaction, and the γ-GTP activity value was determined by the same procedure as in Example 1. Furthermore, using R1-B and R2-B, the γ-GTP activity value was determined in the same manner as above.

Note 1: The value in parentheses in the table is the final concentration during the reaction, and the final concentration during the reaction of CD is 16 mM.

According to the results in Table 2, the presence of a nonionic surfactant during the reaction increases the activity value. Preferably, if a surfactant with a concentration close to the CD concentration is allowed to coexist, it becomes possible to cancel out the disadvantages due to the addition of CD.

[0020]

[Effect of the invention] Since the present invention is configured as described above,
Conventionally, CD and CD derivatives are used, and a poorly soluble or unstable substance is introduced into the measurement system as a guest to improve solubility, and when these substances are reacted, a surfactant is allowed to coexist. Guest molecules can be easily released from CDs, a high-activity substrate/reaction solution can be formed on the spot, and the action of the substrate molecule can be effectively exerted, and the initial absorbance of the reagent can be reduced. Since it is possible to offset color tone changes resulting from suppression or addition of CDs to the measurement system, it has the effect of improving sensitivity, dynamic range, and measurement accuracy.

[Table 1]

Claims (1)

[Scope of Claims] [Claim 1] Measurement of biological components using a biological component measuring reagent containing cyclodextrins as a solubilizer for substances poorly soluble in water or as a stabilizer for unstable substances. A biological component measuring method characterized by coexisting a nonionic surfactant in the measuring system. 2. The method according to claim 1, wherein α-cyclodextrin or a derivative of α-cyclodextrin is used as the cyclodextrin, and a surfactant having a linear alkyl in its hydrophobic group is used as the surfactant. Method for measuring biological components. 3. The method according to claim 1, wherein β-cyclodextrin or a derivative of β-cyclodextrin is used as the cyclodextrin, and a surfactant having an alkylphenyl group in its hydrophobic group is used as the surfactant. Method for measuring biological components. 4. A reagent kit for measuring biological components comprising at least the following two groups of reagents (A) and (B). (A) A reagent containing cyclodextrins as a solubilizing agent for substances poorly soluble in water or as a stabilizer for unstable substances (B) A reagent containing a nonionic surfactant [Claim 5] ] The method for measuring biological components according to claim 4, wherein α-cyclodextrin or a derivative of α-cyclodextrin is used as the cyclodextrin, and a surfactant having a linear alkyl in its hydrophobic group is used as the surfactant. Reagent kit. 6. The method according to claim 4, wherein β-cyclodextrin or a derivative of β-cyclodextrin is used as the cyclodextrin, and a surfactant having an alkylphenyl group in its hydrophobic group is used as the surfactant. Reagent kit for measuring biological components.