MXPA06010539A - Method of stabilizing oxidizable color-assuming reagent - Google Patents

Abstract

A method of storing/stabilizing an oxidizable color-assuming reagent, especially a leuco dye;and a stabilized reagent obtained thereby. The method of stabilizing an oxidizable color-assuming reagent comprises storing the oxidizable color-assuming reagent in a solution having a pH of 1 to 5.

Description

METHOD TO STABILIZE THE REAGENT FOR THE DEVELOPMENT OF OXIDIZED COLOR TECHNICAL FIELD The present invention relates to a method for stabilizing an oxidizable color development reagent to be used for testing the minor components of a biological sample, and to an oxidizable color development reagent stabilized by the method.

TECHNICAL BACKGROUND The testing of several components contained in a biological sample, such as blood and urine, is essential for the diagnosis of the disease, clarification of the pathological conditions or evaluation of the therapeutic procedures, because said components are believed to be involved in some diseases. For example, there are methods that have been developed to test many varieties of minor components, such as blood cholesterol, triglycerides, glucose, uric acid, phospholipids, bile acid, and monoamine oxidase. These methods are used in the diagnosis of some diseases. Among the methods for testing serum components is the enzymatic method in which an enzyme that specifically acts on a The target component becomes active, and its resulting product is tested for the determination of the quantity of the target component. This method is widely used. Among others, it is common to use a method wherein an oxidase that specifically acts on a target component is made to act on the component, to thereby generate hydrogen peroxide, and subsequently, a color development system is established through the contacting the generated hydrogen peroxide with an oxidizable color developing reagent (ie, a reagent that develops color when oxidized), and peroxidase (POD), thereby causing the reagent to develop color; and the amount of the objective component is determined through the colorimetric analysis of the color thus developed. Examples of oxidizable color development reagents employed in said enzymatic method include Trinder reagents, which is a phenolic, aniline, or toluidine chromogen, and forms a dye through oxidation-condensation with a coupler (e.g. aminotipyrine (4-AA) or 3-methyI-2-benzothiazolinonehydrazone (MBTH) in the presence of POD However, the color development system associated with said oxidizable color development reagent has some drawbacks, such as a low sensitivity for the quantification of minor components and their tendency to be affected by changes in the absorption spectrum attributed to hemoglobin, bilirubin, etc., contained in a sample to be tested In recent years, many reports have been published in order to overcome said drawbacks, with respect to oxidizable color development reagents including leuco dye of triphenylmethane and a leuco dye of diphenylnaphthylmethane, which directly develops the color through oxidation in the presence of POD (see, for example, Patent Document 1). In addition, the triphenylmethane compounds are known to improve the low water solubility of the leuco dye (Patent Document 2). The leuco dyes are highly sensitive in tests, and in this way they are very useful compounds for the quantification of minor compounds. However, leuco dyes still have the problem that their storage stability is poor, therefore causing an unspecified, non-specific color development that occurs over time. Patent Document 1: JP-A-62-93261 Patent Document 2: JP-A-3-206896 BRIEF DESCRIPTION OF THE INVENTION Problems to be solved through the invention Accordingly, an object of the present invention is to provide a method for stably storing an oxidizable color developing reagent, in particular, a leuco dye. Another object of the present invention is to provide a stabilized reagent through said method.

Means for solving the problems In view of the foregoing, the present inventors conducted extensive studies, and found that when an oxidizable color developing reagent is stored in a solution having a pH of 1 to 5, the oxidizable color development reagent It can be stably stored for a long period. In this way, the present invention was completed on the basis of this finding. Accordingly, the present invention provides a method for stabilizing an oxidizable color development reagent, which comprises storing the oxidizable color development reagent in a solution having a pH of 1 to 5. The present invention also provides a solution of oxidizable color development reagent having a pH of 1 to 5.

Effect of the invention According to the stabilization method of the present invention, an oxidizable color developing reagent can be stably stored in a solution for a long period. In addition, the use of an oxidizable color development reagent solution of the present invention enables the highly sensitive assay of a minor component of a biological sample. Accordingly, the oxidizable color development reagent solution of the present invention is very useful in the field of clinical examination.

DETAILED DESCRIPTION OF THE INVENTION The oxidizable color development reagent solution of the present invention can be used in any oxidation substance quantification method that utilizes an oxidizable color development reagent as a color development component. Examples of the oxidation substance include hydrogen peroxide. The oxidizable color development reagent solution of the present invention is particularly useful for testing minor components of a biological sample, wherein an oxidase is made to act on a substrate or a substance generated through an enzymatic reaction, and the hydrogen peroxide thus generated is quantified. No particular limitation is imposed on the minor components contained in a biological and quantifiable sample through the use of the oxidizable color development reagent solution of the present invention. In this way, any biological component that can be assayed through the quantification of hydrogen peroxide generated as a result of the enzymatic reaction can become a measurement target of the present invention. Examples of said component include glycated proteins, glycated peptides, glycated amino acids, cholesterol, glucose, glycerin, triglyceride, free fatty acids, uric acid, phospholipids, sialic acid, bile acid, pyruvic acid, inorganic phosphorus, creatinine, creatine, GOT, GPT , monoamine oxidase, guanase, and cholinesterase, etc. No particular limitation is imposed on the oxidizable color development reagent usable in the present invention. Examples of the reagent include a combination of 3-methyl-2-benzothiazolinonehydrazone (MBTH) and an aniline compound; Z, Z'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); leuco dyes; benzidine derivatives, o-tolidine derivatives, triallylimidazole derivatives, and o-phenylenediamine derivatives, etc. Of these reagents, leuco dyes are preferred. Examples of leuco dyes include triphenylmethane derivatives, phenothiazine derivatives, and diphenylamine derivatives, etc. Examples of triphenylmethane derivatives that can be used include highly water soluble compounds described in JP-A-3-206896 and JP-A-6-197795, examples of phenothiazine derivatives include the compounds described in JP-B-60- 33479, and examples of the diphenylamine derivatives include the compounds described in JP-B-60-33479 and JP-A-62-93261. Of these compounds, the preferred ones are malachite green leuco, crystal violet leuco, the sodium salt of N- (carboxymethyl-aminocarbonyl) -4,4'-bs (di-methalamino) -diphenylamine ( DA-64; Wako Puré Chemical Industries Ltd.), sodium salt of 10- (carboxymethyl-aminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA-67: Wako Puré Chemical Industries Ltd.), 10- (N -methylcarbamoi!) - 3,7-bis (dimethylamino) -10H-phenothiazine (MCDP: product of Dojindo laboratories), and N, N, N ', N', N ", N" -hexa-3-sulfopropyl-4 , 4 ', 4"-triaminotriphenylmethane (TPM-PS: product of Dojindo laboratories), etc. these dyes, TPM-PS, DA-64, DA-67, and MCDP are more preferred, with TPM-PS and MCDP being even more preferred. Other usable dyes include diaminobenzidine, tetramethylbenzidine, hydroxyphenylpropionic acid, and orthophenylenediamine, etc. In order to modify the pH, any suitable substance can be used as long as it can achieve an acidic pH. For example, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid can be used; and organic acids such as glycine, phthalic acid, maleic acid, citric acid, succinic acid, oxalic acid, tartaric acid, acetic acid, and lactic acid. No particular limitation has been imposed on the concentration of the inorganic or organic acids, but the concentration is preferably 0.0001 to 1, 000 mM, particularly preferably 0.01 to 1, 000 mM. The pH can be from 1 to 5, but the pH from 1 to 4 is particularly preferred. No particular limitation has been imposed on the concentration of the oxidizable color development reagent contained in an oxidizable color development reagent solution. Preferably, the concentration is from 0.001 to 100 mM, more preferably from 0.001 to 50 mM. The oxidizable color development reagent solution of the present invention may also contain, for example, an anionic or nonionic surfactant, each having a polyoxyethylene structure; an enzyme to treat contaminants in the blood sample, a reaction control agent; a stabilizer; a protein such as albumin; a salt such as sodium chloride, potassium chloride or potassium ferrocyanide; an amino acid such as glycine, lysine, alanine, aspartic acid, or glutamic acid; a tetrazolium salt to avoid the effects of a reduction of the substance; an antibiotic; an antiseptic agent such as sodium azide or boric acid, or a cationic surfactant. The amount of said additive may be appropriately determined with reference to known enzymatic quantification methods that utilize an oxidizable color development reagent. The oxidizable color development reagent solution of the present invention can be provided as contained in, for example, a glass container or a plastic container. The containers are preferably protected from light.

EXAMPLES The present invention will now be described in greater detail with reference to the examples, which should not be construed as limiting the invention.

EXAMPLE 1 Stabilization test 1 for TPM-PS TPM-PS was dissolved in each of the aqueous solutions described below to achieve a concentration of 60 μM, and stored at 37 ° C. Subsequently, the absorbance at 600 nm was measured by means of an automated analyzer (Model 7150, product of Hitachi, Ltd.). Table 1 shows the absorbance data measured at points in time of 0 (start), storage for 2 weeks, and storage for 3 weeks.

TABLE 1 PB-K: potassium phosphate solution.

As is evident from Table 1, the non-specific color development of TPM-PS was effectively suppressed in aqueous solutions of pH 1 to 5, indicating that TPM-PS is stable.

EXAMPLE 2 Stabilization test 2 for TPM-PS TPM-PS was dissolved in each of the aqueous solutions described below to achieve a concentration of 100 μM, and the absorbance was measured at 600 nm. After each sample was stored at 25 ° C for 10 days, the absorbance was remeasured at 600 nm. Table 2 shows the difference between the absorbance measured immediately after sample preparation and that measured after 10 days of storage (referred to as "variation in 10 days (OD)").

TABLE 2 As is evident from Table 2, the variation of absorbance of TPM-PS remains on a small scale when stored in an aqueous solution with a pH of 1 to 5, indicated that TPM-PS is stably stored as a result.

EXAMPLE 3 Stability of MCDP MCDP was dissolved in methanol to achieve a concentration of 4 mM, and the resulting MCDP-methanol was dissolved in each of the aqueous solutions described below supplemented with 0.1% Triton X-100, to achieve a concentration of 100 μm. The solutions were stored at 37 ° C for 24 hours. Subsequently, the absorbance at 600 nM was measured. The results are shown in table 3.

TABLE 3 As is evident from Table 3, the variation of the absorbance of MCDP is too small, and the non-specific color development of MCDP is effectively suppressed when stored in an aqueous solution of pH from 1 to 5, indicating that MCDP is Can store stably.

Claims (7)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for stabilizing an oxidizable color development reagent, which comprises storing an oxidizable color developing reagent in a pH solution of 1 to 5.

2. The method according to claim 1, further characterized in that the reagent Oxidizable color development is a leuco dye selected from a leuco dye of triphenylmethane, a leuco dye of phenothiazine, and a leuco diphenylamine dye.

3. The method according to claim 2, further characterized in that the leuco dye of triphenylmethane is N, N, N ', N', N ", N" -hexa-3-sulfopropyl-4,4 ', 4" -triaminotriphenylmethane 4.- The method according to claim 2, further characterized in that the leuco dye phenothiazine is 10- (N-methylcarbamoyl) -3,7-bis (dimethylamino) -1 OH-phenothiazine or 10- (carboxymethyl) -aminocarbonyl) -3,7-bis (dimethylamino) phenothiazine 5. The method according to any of claims 1 to 4, further characterized in that the pH solution of 1 to 5 contains one or more species selected from among acid hydrochloric acid, sulfuric acid, phosphoric acid, and organic acids 6. The method according to claim 5, further characterized in that the organic acid is maleic acid or citric acid. 7. A solution of an oxidizable color development reagent, characterized in that the solution has a pH of 1 to 5.