JP3055036B2 - Biological component measuring method and measuring reagent kit - Google Patents

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, a biological component is measured using a reagent in which cyclodextrin (hereinafter, also referred to as CD) is used as a solubilizer for a substance which is hardly soluble in water or as a stabilizer for an unstable substance. Sometimes, by coexisting a nonionic surfactant in the measurement system, a biological component measuring method and a biological component devised to promote the release of the poorly water-soluble substance or the unstable substance into an aqueous solution. The present invention relates to a measurement reagent kit.

[0002]

2. Description of the Related Art Generally, aqueous reagents for measuring biological components used for biochemical measurements such as enzyme reactions include color-forming substances such as enzyme reaction substrates, coenzymes, inhibitors, and dyes. It is necessary to uniformly dissolve (contain) a substance that is hardly soluble in water or an unstable substance such as a preservative. There have been various attempts to dissolve these hardly soluble or unstable substances in water at high concentrations, but there is no perfect method.
For this reason, there are substances which cannot be used as biological component measuring reagents or are not widely used. In addition, these poorly water-soluble substances are usually solubilized by a clever method at the time of preparing a biological component measurement reagent, but crystallization, precipitation, hydrolysis, oxidation, etc. are likely to occur over time. Once crystallization or precipitation occurs, the concentration of the substance is reduced, and the function as a highly accurate biological component measuring reagent is lost, thereby losing its value.

[0003] One of the effective methods for dissolving such a poorly soluble substance in a biological component measuring reagent and preventing crystallization or precipitation with the lapse of time has been known as a method which has been conventionally used for a poorly soluble substance in water. A method of coexisting a substance with CD (or CD derivative) or containing it as an inclusion compound has been disclosed (Japanese Patent Application Laid-Open No. 57-74099, Japanese Patent Application Laid-Open No. 60-16 / 1985).
0896, JP-A-61-260900, JP-A-63-260900
129999, Japanese Patent Application No. 63-144717).

In some of these methods, the solubility of the CD itself used is too small to be able to actually dissolve the hardly soluble substance at a high concentration, or the CD derivative is extremely expensive, so Although there are some that cannot be put into practical use at all, there were the following common drawbacks. (1) The substance introduced into the measurement system by improving the solubility using CD and CD derivative is hardly released since the substance is included by CD, and the substance must react (act). It does not show sufficient activity as expected in a must-have situation. That is, even if the hardly soluble or unstable substance is contained in the measurement system at a concentration sufficient as a calculated amount by the solubilizing effect of CD, the effective concentration actually decreases due to the inclusion effect of CD, Will run out. (2) It has been conventionally known that when CDs are used to increase the solubility of a coloring matter, the color tone may be changed (the absorption spectrum may be shifted). This is often caused by the fact that the dye introduced into the measurement system is hardly released because it is included by the CD. This change in color tone is often unfavorable in terms of measurement operation when a clinical test reagent or a general analysis reagent is used, and has been a problem to be solved in a reagent utilizing CD.

[0005] The inventor has previously disclosed in Japanese Patent Application Laid-Open No. Hei 2-2037.
No. 97 discloses a biological component measuring method and a biological component measuring reagent kit in which fatty acids coexist in a measurement system to promote the release of the hardly soluble or unstable substance (guest) from CD. There is a fact. When a fatty acid is used as described above, the reaction system under alkaline conditions has a relatively small molecular weight, so that a reagent having a sufficient molar concentration can be prepared, and the desired effect can be sufficiently exerted. Since the solubility of the fatty acid itself is significantly reduced in the system, there is room for improvement as a guest release promoting substance.

[0006] Then, the inventor further conducted a thorough search for a substance for promoting the release of a guest, and as a result, found that a nonionic surfactant was a substance suitable for this purpose, and completed the present invention. That is, it has been found that if a nonionic surfactant coexists in the final reaction system, it can be used as an effective guest release promoting substance in a reaction system (reagent) in a wide pH range regardless of acidity or alkalinity.

[0007]

The present invention provides the following (1).
To (6). (1) When measuring a biological component using a biological component measuring reagent containing cyclodextrins as a solubilizing agent for a substance which is hardly soluble in water or as a stabilizer for an unstable substance, non-ionic A method for measuring a biological component, comprising coexisting a system surfactant. (2) The living body according to (1), wherein α-cyclodextrin or a derivative of α-cyclodextrin is used as the cyclodextrin, and a surfactant having linear alkyl in the hydrophobic group is used as the surfactant. Component measurement method. (3) The organism according to (1), wherein β-cyclodextrin or a derivative of β-cyclodextrin is used as the cyclodextrin, and a surfactant having an alkylphenyl group in the hydrophobic group is used as the surfactant. Component measurement method. (4) A reagent kit for measuring a biological component comprising at least the following two groups of reagents (A) and (B). (A) a reagent containing cyclodextrins as a solubilizer for a substance that is hardly soluble in water or a stabilizer for an unstable substance; (B) a reagent containing a surfactant; (5) a cyclodextrin; using α-cyclodextrin or α-cyclodextrin derivative,
The reagent kit for measuring a biological component according to the above (4), wherein a surfactant having a straight-chain alkyl in the hydrophobic group is used as the surfactant. (6) As a cyclodextrin, β-cyclodextrin or a β-cyclodextrin derivative is used,
The reagent kit for measuring a biological component according to the above (4), wherein a surfactant having an alkylphenyl group in a hydrophobic group portion is used as the surfactant.

In the present invention, a substance which is hardly soluble in water,
Alternatively, when an unstable substance is stably present as an aqueous solution at a high concentration, a desired reagent concentration should be given by using CD or a CD derivative, and the non-ionic surfactant is used in the final measurement system or reaction system. With the agent,
A guest molecule, which has already formed an inclusion complex with CD, undergoes a substitution reaction with a nonionic surfactant molecule, and the guest
In addition to eliminating the adverse effects of the class, such as color change, and increasing the activity concentration of the guest (original active substance), a more effective measurement system and reaction system can be realized by combining reagents. It is.

In the present invention, the substance that is hardly soluble in water or the substance that is unstable refers to relatively low-molecular organic compounds described in the following (a) to (f). In particular, when using a poorly soluble and unstable enzyme substrate such as tyrosine, tryptophan, adenosine, xanthine, and uric acid, the present invention is extremely suitable.Furthermore, nitroaniline and nitrophenols are detected. The configuration of the present invention is also very suitable for the reaction system described above. (A) Substrate for enzyme reaction (A) Poorly soluble amino acids such as phenylalanine, tyrosine, and tryptophan (B) Nucleic acid bases that are poorly soluble in water such as adenine, guanine, thymine, cytosyl, uracil, hypoxanthine, xanthine, uric acid, and inosine Or its metabolites (C) thyroxine, adrenaline, noradrenaline,
Low molecular sparingly soluble hormones such as catecholamines and melatonin (D) Steroid hormones such as testosterone, aldosterone, corticosterone, progesterone and estriol (b) Coenzymes (vitamins): nicotinamide adenine dinucleotide, flavin adenine dinucleotide And non-nucleotide coenzymes such as pyridoxal phosphate, biotin, folic acid, lipoic acid, retinal, carotene, α-tocopherol, vitamin K3, ubiquinone, and vitamin D. Hydroquinone, catechol, pyrogallol, vanillin and the like and derivatives thereof (B) Nitroaniline, acid anilide derivative of chloronitroaniline, nitrophenol, ester bond derivative of chloronitrophenol or ether (C) Phenolphthalein, acridine, phenazine, fluorescein, rhodamine, naphthalene, pyrene, anthracene, umbelliferone, coumarin derivatives and hapten labeled with them (d) inhibitors: 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, bathocuproin (B) lipids such as triglycerides and cholesterol

In the present invention, CDs are α-,
unmodified CD such as β-, γ-CD, maltosylated α-, β
-CD, so-called branched CD derivatives such as glucosylated α- and β-CD, and CD derivatives having a side chain introduced for improving water solubility, such as hydroxypropyl-CD.

The surfactant used in the present invention is basically composed of a sugar alcohol such as polyoxyethylene or sorbitol whose hydrophilic group is nonionic or a derivative thereof.
The hydrophobic group is composed of an alkyl group having 4 or more unit numbers or an alkylphenyl group, and both residues are covalently bonded by an ether bond or an ester bond (both residues may be a phosphate ester bond via phosphoric acid). It has a structure. When α-CD or a derivative thereof is used for CDs, a nonionic surfactant (for example, Briji series) whose hydrophobic group is a linear alkyl group is used for β-CD or CD- derivatives.
When CD or a derivative thereof is used, the hydrophobic portion is an alkyl group and preferably an alkylphenyl group (for example, T
(Riton-X series) alone or in combination. Further, a plurality of nonionic surfactants may be used respectively. Furthermore, the concentration of the nonionic surfactant to be coexisted, particularly the molecular concentration of the hydrophobic group portion, is sufficient to achieve the effect, but the guest in which the nonionic surfactant molecule is included in the CDs is sufficient. Because of the need to replace the molecule, it is practical to make it at least larger than its guest molarity. Further, it is often preferable to add the hydrophobic group portion of the nonionic surfactant to a level exceeding the molar concentration of the added CDs, since most of the guest molecules can be released to the reaction system. It is desirable to increase the concentration of the nonionic surfactant added as compared with at least the concentration of the CDs or the concentration of the guest molecule, which shows a smaller molar concentration.

The present invention is defined as a biochemical analysis item, γ
-Glutamyltransferase (EC 2.3.2.
The following describes an example of an activity measurement system. It is known that the enzyme activity γ-glutamyltransferase (γ-GTP) using γ-L-glutamyl-p-nitroanilide (γ-GpNA) as a substrate has a Km (Michaelis-Menten constant) of about 1 mM for this substrate. Have been. From this Km value, γ-GT
In order to accurately measure P activity, the concentration of the substrate contained in the reagent needs to be several mM or more. However, pH 4
At a pH other than -5, the solubility can be relatively increased, but since the hydrolysis rate is further increased, it cannot be stored as a reagent at a pH other than the above. pH
At around 4 to neutral, the solubility of γ-GpNA is minimal,
In order to prepare a reagent that can be used practically and to store it at a low temperature since it dissolves in less than mM (4 ° C.), CD (in this case, CD
Improved solubility of itself, for example, maltosylated CD)
Need to be added to increase the solubility. However,
When the substrate solution to which the CD is added is used, there are the following disadvantages. Enzyme activity is lower than expected. This is because, as a problem in the properties of the enzyme itself whose activity is to be measured, the affinity between the enzyme (GT) and the substrate is low, so that the substrate cannot be deprived from the CD inclusion, and the calculation is not sufficient. Despite the fact that the substrate is supplied at a concentration, the enzyme reaction system is not given a substantially high concentration of the substrate. This phenomenon is an inconsistency in a practical system, in which an enzyme having a large Michaelis constant and its substrate were dissolved in a high concentration as an aqueous solution by CD because the substrate was hardly soluble. In particular, when α-CD or its derivative is contained in the CD composition, the absorbance of the reagent blank must be extremely high. This is because a dye molecule having a nitro group and a benzene ring, or a derivative thereof, is included in the CD, causing a spectral shift to the red direction. This is due to the phenomenon of increasing the absorbance of a reagent blank of a reaction system containing a large amount of a substrate, that is, a dye derivative. In particular, the case where CD containing α-CD or a derivative thereof is added to the reagent is remarkable. Then, by coexisting a nonionic surfactant in the reaction system and easily releasing γ-GpNA from CDs, the above-mentioned problems can be solved at once and extremely accurate measurement becomes possible.

[0013]

According to the present invention, when a substance that is hardly soluble in water or an unstable substance is stably present in a high concentration as an aqueous solution, a desired reagent composition is provided by using CD or a CD derivative. In the final measurement system, a nonionic surfactant is allowed to coexist to cause a substitution reaction with the guest, thereby eliminating the influence that the guest has received from the CD, for example, the adverse effect such as a change in the color tone, and the effect of the guest. It increases the concentration.

[0014]

Example 1 <Effects of Surfactants on CD Derivatives> * To confirm the effects of various surfactants on CD derivatives, the following reagents 1 and 2 were prepared, and γ-GTP was prepared by the following operation. Activity values were determined. Reagent 1: 100 mM containing 80 mM glycylglycine
In a Tris-HCl buffer, a nonionic surfactant described in the following (1) was uniformly added at 25 mM to adjust the pH to 8.3, and (1) No surfactant was added (no addition,
Control) (to be referred to as R1-〓, R1-, etc.) in total. 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 X405 (Triton-X405: polyoxyethylene (n @ 40) -pt-octylphenol) Reagent 2: 80 mM Glucosyl-α-CD (G-α-
CD) and a substrate solution containing 20 mM of γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride (hereinafter referred to as R2-), 80 mM of glucosyl-β-CD
(G-β-CD) and 20 mM γ-L-glutamyl-
A substrate solution containing p-nitroanilide (γ-GpNA) hydrochloride (hereinafter referred to as R2-). * Operation: 25 μL of commercially available serum (with a γ-GTP activity value of 47 IU / L according to the standard method) and R1-〓 to R1
After mixing each of 1000 μL and heating at 37 ° C. for 5 minutes, 250 μL each of R 2-or R 2-is added, and the reaction is started. 405 nm wavelength after the start of the reaction
Γ-GTP activity value in each of the reagents is determined from the previously determined molecular extinction coefficient of P-nitroanilide as a reaction product.

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

According to the results shown in Table 1, when the α-isomer is used as the CD, it is particularly preferable to use a nonionic surfactant having a straight-chain alkyl in the hydrophobic group as the nonionic surfactant. In the case where a β-form is used, it is found that those having an alkylphenyl group in the hydrophobic group are particularly excellent. The above results were similar to the results measured using maltosyl-α-CD and maltosyl-β-CD.

[0017]

Example 2 <Study on Addition of Surfactant> * The following reagent (R) was prepared. R1-A: 100 m containing 80 mM glycylglycine
Bridge 35 (straight chain alkyl type) was added to 0,5,10,15,20,2
Those added to give concentrations of 5, 30 and 50 mM. R1-B: 100 m containing 80 mM glycylglycine
Triton X-10 in M Tris-HCl buffer (pH 8.3)
0 (alkylphenyl group type) is converted to 0,5,10,1
Those added to give concentrations of 5, 20, 25, 30, and 50 mM. R2-A: 80 mM glucosyl-α-CD (G-α-CD)
A substrate solution containing CD) and 20 mM of γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride. R2-B: 80 mM glucosyl-β-CD (G-β-
A substrate solution containing CD) and 20 mM of γ-L-glutamyl-p-nitroanilide (γ-GpNA) hydrochloride. * Operation: 25 μL of commercially available serum (with a γ-GTP activity value of 47 IU / L according to the standard method) used in Example 1 and R
After mixing 1000 μL of 1-A and 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 in the same manner as in Example 1 below. Further, using R1-B and R2-B, the γ-GTP activity value was determined in the same manner as in the above operation.

[0018] Note 1: In parentheses in the table, the final concentration during the reaction is 16 mM.

According to the results shown in Table 2, the activity value increases when a nonionic surfactant is present during the reaction. Preferably, if a surfactant having a concentration close to that of the CD is used, the disadvantage can be eliminated by adding the CD.

[0020]

The invention of the present application has been configured as described above.
Conventionally, using a CD and a CD derivative, a poorly soluble substance or an unstable substance is used as a guest to improve solubility and is introduced into a measurement system. When reacting these substances, a surfactant is allowed to coexist. The guest molecules up to that can be easily released from the CDs, and a high-activity substrate / reaction solution can be formed on the spot, so that the action of the substrate molecules can be effectively exerted. Since the change in color tone caused by the suppression and the addition of CDs in the measurement system can be offset, the effect of improving sensitivity and dynamic range and improving measurement accuracy is obtained.

[Table 1]

Claims (6)

(57) [Claims] When a biological component is measured using a reagent for measuring a biological component containing cyclodextrins as a solubilizing agent for a substance that is hardly soluble in water or a stabilizer for an unstable substance, the measurement system is not used. A method for measuring a biological component, comprising coexisting an ionic surfactant. 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 straight-chain alkyl in the hydrophobic group is used as the surfactant. Biological component measurement method. 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. Biological component measurement method. 4. At least two of the following (A) and (B)
A reagent kit for measuring a biological component, comprising a group of reagents. (A) A reagent containing cyclodextrins as a solubilizer for a substance that is hardly soluble in water or a stabilizer for an unstable substance. (B) A reagent containing a nonionic surfactant. 5. The method according to claim 4, wherein α-cyclodextrin or a derivative of α-cyclodextrin is used as the cyclodextrin, and a surfactant having a straight-chain alkyl in the hydrophobic group is used as the surfactant. Reagent kit for measuring biological components. 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 the hydrophobic group is used as the surfactant. Reagent kit for measuring biological components.