JP7061283B2 - How to stabilize leuco-type dye - Google Patents

Description

The present invention relates to a method for stabilizing a color-developing substrate solution and a stabilized color-developing substrate solution used in fields such as clinical examinations. More specifically, the present invention relates to a method for suppressing coloration of a color-developing substrate solution containing a leuco-type dye over time and a color-developing substrate solution in which coloration over time is suppressed.

In the field of clinical examination, a method of detecting trace components by a color-developing reaction using peroxidase is known. For example, in the method for measuring a glycated protein, a color-developing substrate and hydrogen peroxide are usually used as the substrate for peroxidase. Here, the measurement of peroxidase activity is performed by oxidizing the color-developing substrate by the action of peroxidase and hydrogen peroxide in a substrate solution for measuring peroxidase activity (hereinafter referred to as a substrate solution for measurement) containing a color-developing substrate and hydrogen peroxide to develop color. This is done by producing a dye and measuring the absorbance of the produced color dye. As the color-developing substrate, there is a dye called a Trinder reagent or a dye called a leuco-type dye. The Trinder reagent is a dye composed of two types, a coupler such as 4-aminoantipyrine and various Trinder reagents, and is characterized by being a dye stable to light and heat. Regarding sensitivity, it reacts with two molecules of hydrogen peroxide. On the other hand, leuco-type dyes are 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA-67) and N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenyl. High-sensitivity measurement is possible because it reacts with one molecule of hydrogen peroxide due to amine (DA-64), etc., but it is unstable to light and heat, so there are problems such as a high blank before measurement. There are various studies on the suppression of self-coloring of leuco-type dyes.

So far, a method of adding a reducing agent has been known as a method of stabilizing a substrate solution containing a color-developing substrate used for measuring peroxidase activity (Patent Document 1). Further, as a method for reducing the non-specific color reaction of the leuco-type dye, 1) a dye having a maximum absorption wavelength in the range of 400 nm to 550 nm and a dye different from the leuco-type dye that does not react with hydrogen peroxide, and 2). A method for coexisting cyclodextrins is known (Patent Document 2). However, the technique for suppressing the coloring of the color-developing substrate liquid containing the leuco-type dye is not yet sufficient.

Japanese Patent No. 3289280 Japanese Patent No. 4697809

As described above, the color-developing substrate solution containing the leuco-type dye is extremely unstable and is easily oxidized and colored, so that the substrate solution containing the color-developing substrate cannot be stored for a long period of time. From such a problem, stabilization of a substrate liquid (particularly a substrate liquid for measurement) containing a color-developing substrate of a leuco-type dye is strongly desired, but a method that is sufficiently satisfactory as a stabilization method is not known. The present invention has been made to eliminate the above-mentioned drawbacks of the prior art, and improves the stability of the color-developing substrate solution containing the leuco-type dye, especially the substrate solution for measurement containing the leuco-type dye, and suppresses coloring. It is an object of the present invention to provide a substrate solution which is stabilized and whose coloring is suppressed.

As a result of diligent studies on the stabilization of the color-developing substrate liquid containing the leuco-type dye, the present inventors have obtained the self-coloring of the leuco-type dye by adding sulfite and a nonionic surfactant as stabilizers. It was found that it was suppressed and the color-developing substrate solution could be stabilized. That is, the present invention is to add a sulfite and a nonionic surfactant to a color-developing substrate solution containing a leuco-type dye as a color-developing substrate. The stabilization method of the present invention can be used, for example, for measuring glycated protein using a color-developing substrate solution containing a leuco-type dye, but is not limited to measuring glycated protein, and is used in other inspection methods using the color-developing substrate. Also, it can be widely used as a method for stabilizing a color-developing substrate solution.

That is, the present invention has the following configuration.
(Item 1) A method for suppressing coloration of a color-developing substrate liquid containing a leuco-type dye, which comprises coexistence of a leuco-type dye with a sulfite and a nonionic surfactant.
(Item 2) The method according to Item 1, wherein the sulfite is sodium sulfite.
Item 3. The method according to Item 1 or 2, wherein the nonionic surfactant is a polyoxyethylene-based nonionic surfactant.
(Item 4) The method according to Item 3, wherein the polyoxyethylene-based nonionic surfactant is a polyoxyethylene alkyl ether.
Item 5. The method according to Item 3 or 4, wherein the polyoxyethylene-based nonionic surfactant is polyoxyethylene lauryl ether.
(Item 6) The method according to any one of Items 1 to 5, wherein the leuco-type dye is 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium.
(Item 7) The method according to any one of Items 1 to 6, which has a pH of 4 to 9.
(Item 8) A color-developing substrate liquid containing a leuco-type dye, a sulfite, and a nonionic surfactant.

According to the present invention, by using a sulfite and a nonionic surfactant as a stabilizer of the color-developing substrate solution containing the leuco-type dye, non-enzymatic oxidation of the color-developing substrate solution is suppressed, so that the color-developing substrate is suppressed. The liquid can be stabilized, and the color-developing substrate liquid can be stably stored for a long period of time. Therefore, according to the present invention, it is possible to provide a color-developing substrate liquid containing a leuco-type dye that is stable for a long period of time and has high accuracy.

In the present invention having the above structure, examples of the leuco-type dye include a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative and the like. Specifically, as a triphenylmethane derivative, 4,4'-benzylenebis (N, N-dimethylaniline) and the like; as a phenothiazine derivative, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino). Phenothiazine salts (DA-67), 10- (methylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine, 10- (N-methylcarbamoyl) -3-dimethylamino-7-hydroxy-10H-phenothiazine, etc.; Examples of the diphenylamine derivative include 4,4'-bis (dimethylamino) diphenylamine, N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine salt (DA-64); 1- (ethylaminothio). Examples thereof include carbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole. Here, the salt is not particularly limited, and examples thereof include sodium salt, potassium salt, calcium salt and the like. It is desirable that the leuco-type dye used in the present invention is less susceptible to color development inhibition by a reducing agent such as sulfite added as a stabilizer, or has a stabilizing effect even with a low concentration reducing agent. .. When the method of the present invention is used in a peroxidase measurement system, the molar extinction coefficient is high, the maximum absorption wavelength is on the high wavelength side, the wavelengths of coexisting substances in blood can be avoided, and the wavelength of the dye can be easily detected. For this reason, phenothiazine derivative-based or diphenylamine derivative-based leuco-type dyes are preferable, and phenothiazine derivative-based leuco-type dyes are more preferable, and 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt (DA). -67) is particularly preferable.

As the sulfite used in the present invention, any sulfite can be used as long as it does not inhibit the desired reaction (for example, the enzymatic reaction of peroxidase), but sodium salt, potassium salt and the like are preferable. Metal salts are mentioned, and two or more kinds of these sulfites may be used in combination. A particularly preferred example of sulfite is sodium sulfite.

As the nonionic surfactant used in the present invention, any nonionic surfactant can be used as long as it does not inhibit the desired reaction (for example, the enzymatic reaction of peroxidase), but it is preferable. The HLB value of 12 to 20 is used. The structure thereof is not particularly limited, but a compound selected from the group consisting of polyoxyethylene-based nonionic surfactants is preferable. Among them, polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene myristel ether, and polyoxyethylene octyldodecyl ether are preferable. May be used in combination of two or more. A particularly preferred example of a nonionic surfactant is polyoxyethylene lauryl ether.

The present invention stabilizes a color-developing substrate liquid containing a leuco-type dye by using a sulfite and a nonionic surfactant in combination, and suppresses coloring over time. For example, the color-developing substrate of the leuco-type dye is purified water or an appropriate buffer (for example, Good buffer, carbonate buffer, phosphate buffer, citrate buffer, acetate buffer, borate buffer, etc., tartrate buffer). Etc.), etc.), the desired purpose is achieved by adding sulfite and a nonionic buffer to a solution dissolved in a solvent, adding other additive components as necessary, and adjusting the pH as necessary. Will be done. The order of addition when preparing the color-developing substrate solution of the present invention is not particularly limited, and a leuco-type dye may be added to a solution containing a sulfite and a nonionic surfactant, or the leuco-type dye and the leuco-type dye and the like. Sulfites may be added to the solution containing the nonionic surfactant. The color-developing substrate liquid of the present invention is the substrate liquid thus prepared, and can be significantly stabilized by the coexistence of sulfites and nonionic surfactant. The amount of the sulfite and the nonionic surfactant added is appropriately adjusted depending on the type and amount of the leuco-type dye, the type of the sulfite and the nonionic surfactant, the amount of dissolved oxygen, the desired stability and the like.

In certain embodiments, the methods of the invention are suitably used for the measurement of saccharified proteins. When the present invention is applied to the stabilization of a color-developing substrate solution used for measuring a glycated protein, the present invention can be carried out in various embodiments. For example, a method of preparing and storing a color-developing substrate solution containing a leuco-type dye together with a sulfite and a nonionic surfactant, and adding an enzyme at the time of use to prepare a substrate solution for measurement; a leuco-type dye from the beginning. , A method of preparing and storing a color-developing substrate solution for measurement containing a sulfite and a nonionic surfactant together with an enzyme. From the viewpoint of reliably avoiding the possibility that enzymes (peroxidase, etc.) will react with leuco-type dyes, etc. during storage and enabling more accurate measurement, the measurement reagents are prepared in two or more parts, while It is preferable that a leuco-type dye, a sulfite, and a nonionic surfactant coexist in the reagent of the above, and the other reagent contains an enzyme (peroxidase or the like).

The concentration of the color-developing substrate in each of the above-mentioned color-developing substrate solutions can be appropriately adjusted, but usually, the concentration of the leuco-type dye in the color-developing substrate solution is about 0.001 to 1 mM, preferably about 0.005 to 0.5 mM. , More preferably about 0.01 to 0.1 mM.

The amount of sulfite added is the type and concentration of sulfite and nonionic surfactant, the type and concentration of leuco-type pigment, the type and concentration of other compounding components (for example, enzymes, etc.), and the desired stability and color development inhibition. Although it is appropriately adjusted depending on the degree and the like, the sulfite concentration in the color-developing substrate solution is usually about 0.01 to 3 mM, preferably about 0.05 to 2 mM, and more preferably about 0.1 to 1 mM. .. If the amount of sulfites to be added to the biological component measurement reagent is too large, the color development of the leuco-type dye may be inhibited. However, by setting the above range, the present invention suppresses the problem of such a decrease in color development intensity. The effect of can be fully obtained.

The amount of nonionic surfactant added is also the type and concentration of sulfite and nonionic surfactant, the type and concentration of leuco-type dye, the type and concentration of other ingredients (eg, enzymes, etc.), and the desired stability. Although it is appropriately adjusted depending on the nature and the like, the concentration of the nonionic surfactant in the color-developing substrate solution is usually adjusted to be about 0.1 to 12 w / v%, preferably 0.5 to 10 w / v. %, More preferably 1 to 8 w / v%. Within the above range, the effect of the present invention can be sufficiently obtained while suppressing the influence on other compounding components.

More specifically with the preferred sulfite and nonionic surfactants, sodium sulfite and polyoxyethylene lauryl ether, for example, sodium sulfite is 0.05-2 mM and polyoxyethylene lauryl ether is 0.5-. The concentration is adjusted to 10 w / v%, preferably about 0.1 to 1 mM for sodium sulfite and about 1 to 8 w / v% for polyoxyethylene lauryl ether.

When the color-developing substrate solution is prepared according to the present invention, the ratio of the leuco-type dye to the sulfite is not particularly limited as long as the effect of the present invention is exhibited without inhibiting the desired reaction (for example, the enzymatic reaction of peroxidase). As an example, the concentration of leuco-type dye is preferably 0.1 mM, the concentration of sulfite is preferably about 0.01 to 3 mM, and the concentration of sulfite is preferably about 0.1 to 2 mM. , The concentration of sulfites is more preferably about 0.5 to 1.5 mM.

When preparing a color-developing substrate solution according to the present invention, the ratio of the leuco-type dye to the nonionic surfactant exerts the effect of the present invention without inhibiting the desired reaction (for example, the enzymatic reaction of peroxidase). The present invention is not particularly limited, but as an example, the concentration of the leuco-type dye is preferably 0.1 mM, whereas the concentration of the nonionic surfactant is preferably about 0.1 to 12 w / v%, and the nonionic surfactant is used. The concentration of the active agent is preferably about 0.5 to 10 w / v%, and the concentration of the nonionic surfactant is more preferably about 1 to 8 w / v%.

Further, the properties of the color-developing substrate liquid of the present invention are usually adjusted to a pH range of about 4 to 9, more preferably about pH 6 to 8. The pH of the color-developing substrate solution can be adjusted, for example, by adding a buffer solution as appropriate.

When the color-developing substrate solution of the present invention is a liquid reagent used for biochemical analysis, it can be prepared, for example, by dissolving, suspending or dispersing a sulfite, a nonionic surfactant and a leuco-type dye in a solvent. Purified water, an appropriate buffer solution, or the like is used as the solvent, but it is preferable to use a buffer solution. As the buffer solution, any buffer solution can be used as long as it does not inhibit the coexisting enzyme activity. For example, Good buffer solution, carbon dioxide buffer solution, phosphate buffer solution, citrate buffer solution, acetate buffer solution can be used. , Borate buffer, etc., tartrate buffer, etc., and Good buffer is particularly preferable. Each substrate solution thus prepared is preferably stored in a cool and dark place.

The Good's buffer used in the present invention is not particularly limited as long as it does not suppress the subsequent enzymatic reaction, and any Good's buffer known in the art can be widely used. Preferably 2-morpholinoetan sulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), 4- (2-hydroxyethyl) -1-piperazine ethane at concentrations of 10-200 mM. Sulfonic acid (HEPES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 3-morpholinopropanesulfonic acid (MOPS) and the like are used.

In addition to the leuco-type dye, sulfites, and nonionic surfactant, the color-developing substrate liquid of the present invention may further contain any component well known in the art, if necessary. For example, enzymes, preservatives, antioxidants, salts, and stabilizers other than sulfites and nonionic surfactants used in the reaction system for measuring the components can be mentioned.

The enzyme that can be blended in the color-developing substrate liquid of the present invention is not particularly limited, and any enzyme known in the art can be blended. For example, in the case of a color-developing substrate solution used for measuring glycated protein, a protease, peroxidase, and glycated amino acid oxidase can be blended.

The protease that can be used in the present invention is not particularly limited, and aminopeptidase, dipeptidase, dipeptidylpeptidase, trypeptidylpeptidase, peptidyldipeptidase, serinecarboxypeptidase, prolinecarboxypeptidase, alaninecarboxypeptidase, metalloprotease, Examples thereof include cysteine carboxypeptidase, omegapeptidase, serine endopeptidase, cysteine protease, aspartate protease, neutral protease, threonine endopeptidase and the like.

The peroxidase that can be used in the present invention is not particularly limited as long as it can generate a coloring dye by contacting hydrogen peroxide with a coloring substrate, but a suitable one is derived from horseradish, a microorganism, or the like. There is something to do. Of these, peroxidase derived from horseradish is preferable. The peroxidase is commercially available in high purity and low price.

The glycated amino acid oxidase that can be used in the present invention means an enzyme that can catalyze a reaction that oxidizes an amadori compound to generate hydrogen peroxide. The saccharified amino acid oxidase may be referred to as fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosylamine oxidase, amadriase, ketoamine oxidase, saccharified hexapeptide oxidase, saccharified hemoglobin oxidase, etc. Can be used.

The preservative that can be blended in the color-developing substrate liquid of the present invention is not particularly limited, and any preservative known in the art can be blended. For example, specific examples of preservatives include various Proclin (registered trademark, Spellco), azides, chelating agents, antibacterial agents and the like. Examples of the chelating agent include ethylenediaminetetraacetic acid and salts thereof. Examples of the antibacterial agent include gentamicin, kanamycin, chloramphenicol, piperacillin, imidazolidinylurea and the like. By blending an antiseptic, there is an advantage that the color-developing substrate liquid can be prevented from spoiling.

When the preservative is blended in the color-developing substrate liquid of the present invention, the concentration thereof is not particularly limited, but for example, the concentration of the preservative in the color-developing substrate liquid is about 0.005 to 1 w / v%, preferably 0.01 to. It is preferable to set it to about 0.1 w / v%. Further, the color-developing substrate liquid of the present invention may be in an embodiment that does not substantially contain a preservative. Here, "substantially free" means that the concentration of the preservative in the color-developing substrate liquid is less than 0.005 w / v%.

When salts are blended in the color-developing substrate solution of the present invention, the type and concentration thereof are not particularly limited, but sodium chloride, sodium acetate, sodium phosphate, sodium hydroxide, potassium chloride, potassium acetate, potassium phosphate, potassium hydroxide. , Aluminum chloride, aluminum acetate, aluminum phosphate, aluminum hydroxide, calcium chloride, calcium acetate, calcium phosphate, calcium hydroxide and the like. By blending such salts, a more stable color-developing substrate solution can be obtained.

In the present invention, ferrocyanides such as potassium ferrocyanide and nitrites such as sodium nitrite may be used as necessary (for the purpose of avoiding the influence of reducing substances derived from a sample, modifying hemoglobin, promoting the reaction of proteases, etc.). May be added.

When the method of the present invention is applied to the measurement of glycated protein, the operation can be performed in the same manner as the conventional method. For example, after mixing a sample containing glycated protein and an enzyme solution containing an enzyme for measuring glycated protein, The substrate solution for measurement prepared by the above method is mixed and reacted at a predetermined temperature (usually about 37 ° C.) for a predetermined time (usually about 10 minutes). Then, by measuring the absorbance, the reaction amount of the color-developing substrate can be determined. Further, the concentration (amount) of the substance to be measured in the sample can be obtained by comparing with the calibration curve prepared in advance based on the obtained amount of change in absorbance.

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

[Example 1] Evaluation of stability of leuco-type dye A 50 mM PIPES solution containing 0.06 mM DA-67 (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium) as a leuco-type dye. A solution containing 0.5 mM / L of sodium sulfite as a sulfite salt and 10 g / L of polyoxyethylene lauryl ether as a nonionic surfactant was stored at 4 ° C. for 5 or 10 days at (pH 6.3). As a comparative example, a solution in which only sulfite is added to the PIPES solution (pH 6.3) containing DA-67, a solution in which only a nonionic surfactant is added, and a sulfite and a nonionic surfactant are used. A solution containing neither was prepared and stored under the same conditions. The degree of coloration of the leuco-type dye solution itself before and after storage was determined by measuring the absorbance of these solutions at 660 nm immediately after preparation and after storage at 4 ° C. for 5 or 10 days. The obtained absorbance is shown in Table 1.

As can be seen from Table 1, the initial absorbance of the DA-67 alone solution in the absence of the addition of sulfite and nonionic surfactant was 53.7 mAbs 4 days after storage at 4 ° C for 5 days. When sodium sulfite or polyoxyethylene lauryl ether was added, the color development intensity was small. Furthermore, it was clarified that by adding both sodium sulfite and polyoxyethylene lauryl ether, there was almost no change in the absorbance during storage, and the solution containing the color-developing substrate could be stably stored. Further, when the product was stored at 4 ° C. for 10 days, a high degree of color-suppressing effect was observed in the color-developing substrate solution containing DA-67 by using sodium sulfite and polyoxyethylene lauryl ether in combination.

INDUSTRIAL APPLICABILITY The present invention can suppress non-specific color development over time in a color-developing substrate solution using a leuco-type dye, and is suitably used in various inspection methods using the color-developing substrate solution.

Claims (8)

10- (Carboxamide Aminocarbonyl) -3 , characterized by the coexistence of 10- (Carboxamide Aminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium with sulfite and polyoxyethylene alkyl ether . , A method for suppressing coloration of a color-developing substrate solution containing 7-bis (dimethylamino) phenothiazine sodium . The method according to claim 1, wherein the sulfite is sodium sulfite. The method according to claim 1 or 2 , wherein the polyoxyethylene alkyl ether is a polyoxyethylene lauryl ether. The method according to any one of claims 1 to 3 , wherein the pH is 4 to 9. A color-developing substrate solution containing 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium , sulfites and polyoxyethylene alkyl ether . The color-developing substrate liquid according to claim 5, wherein the sulfite is sodium sulfite. The color-developing substrate liquid according to claim 5 or 6, wherein the polyoxyethylene alkyl ether is a polyoxyethylene lauryl ether. The color-developing substrate liquid according to any one of claims 5 to 7, which has a pH of 4 to 9.