JP2008197077A - Method and reagent for measuring object substance to be measured in sample, method for inhibiting nonspecific coloring, and nonspecific coloring inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method and a measuring reagent capable of accurately measuring a concentration of an object substance to be measured in a sample by inhibiting influences of various interfering substances such as hemoglobin, ascorbic acid and the like contained in the sample, in measuring the object substance to be measured in the sample by using a tetrazolium compound. <P>SOLUTION: In measuring the object substance to be measured in the sample by using the tetrazolium compound, the method and the reagent for measuring the object substance to be measured in the sample are characterized in that at least one selected from a group consisting of Trinder's reagents, superoxide dismutase and 1,2-dihydroxy-3,5-benzenedisulfonic acid or its salt is put or contained in a measurement reaction liquid or the measuring reagent, as a nonspecific coloring inhibitor for the tetrazolium compound. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method and a reagent for measuring a substance to be measured in a sample using a tetrazolium compound, a method for suppressing nonspecific color development of a tetrazolium compound, and a nonspecific color development inhibitor for a tetrazolium compound.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

Measuring a substance to be measured in a sample such as blood and observing the fluctuation is indispensable for diagnosis, treatment, early detection and prevention of diseases, and various measurement methods have been developed.
As one of such measurement methods, an oxidation reaction is caused between the electron acceptor and the substance to be measured by an enzyme, etc., and the reductant of the generated electron acceptor is measured, and the measurement object contained in the biological sample. The method of measuring a substance can be mentioned.
For example, nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) is used as an electron acceptor, and reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide, which is a reduced form thereof, is used. An example is a method in which an electron carrier such as diaphorase or 1-methoxy-5-methylphenazinium methylsulfate and a tetrazolium compound are allowed to act on nucleotide phosphate (NADPH), and the resulting formazan dye is colorimetrically measured.

  However, it is also known that the measurement of a substance to be measured in a sample using the tetrazolium compound is easily affected by various interfering substances such as ascorbic acid contained in the sample. That is, the tetrazolium compound is reduced by various interfering substances contained in the sample, and even if NADH or NADPH is not present, a formazan dye is generated, and the measurement value of the measurement target substance in the sample has a positive error. It was known to give (see, for example, Patent Document 1).

  In addition, as a method of suppressing the influence of the interfering substance in the sample, for example, a method of suppressing the influence of ascorbic acid with ascorbic acid oxidase (for example, see Patent Document 2) has been proposed.

  JP-A-64-45374   JP-A-56-39198

Accordingly, an object of the present invention is to suppress the influence of various interfering substances such as hemoglobin and ascorbic acid contained in a sample in measurement of the measurement target substance in a sample using a tetrazolium compound, and To provide a measuring method and a measuring reagent capable of accurately measuring the concentration.
Moreover, the subject of this invention is providing the method of suppressing the nonspecific color development of a tetrazolium compound, and providing the nonspecific color development inhibitor of a tetrazolium compound.

In the measurement of a measurement target substance in a sample using a tetrazolium compound, the present inventor causes non-specific color development of the tetrazolium compound due to hemoglobin contained in the sample, resulting in a positive error in the measurement value of the measurement target substance in the sample. I found out that
In addition, when measuring the measurement target substance in a sample using a tetrazolium compound, the present inventor, even when ascorbic acid oxidase is present in the measurement reagent, the non-specific tetrazolium compound is detected by the ascorbic acid in the sample. It was found that color development occurs and gives a positive error to the measured value of the substance to be measured in the sample. That is, it has been found that in the measurement of a measurement target substance in a sample using a tetrazolium compound, the influence of ascorbic acid is not sufficiently suppressed even if ascorbate oxidase is present in the measurement reagent.
Furthermore, the present inventor, in the measurement of the measurement target substance in the sample using the tetrazolium compound, nonspecific color development of the tetrazolium compound occurs due to various blood components other than hemoglobin and ascorbic acid in the sample, It was found that a positive error was given to the measured value of the substance to be measured.

  As a result of intensive studies aimed at solving the above problems, the present inventor, as a result of measurement of a measurement target substance in a sample using a tetrazolium compound, Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3, The presence of one or more substances selected from 5-benzenedisulfonic acid or its salt in the measurement reaction solution suppresses the influence of various interfering substances such as hemoglobin and ascorbic acid in the sample, The present inventors have found that the concentration of the substance to be measured can be accurately measured, and have completed the present invention.

That is, the present invention provides the following inventions.
(1) In the method for measuring a substance to be measured in a sample using a tetrazolium compound, at least one selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof A method for measuring a substance to be measured in a sample, wherein the substance is present in a measurement reaction solution.
(2) One or more substances selected from a Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof are present as a non-specific color inhibitor of a tetrazolium compound. The method for measuring a substance to be measured in the sample according to (1).
(3) One or more kinds of reagents selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof as a measurement reagent for a substance to be measured in a sample using a tetrazolium compound A reagent for measuring a substance to be measured in a sample, comprising a substance.
(4) One or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof are contained as a non-specific color inhibitor for tetrazolium compounds. The reagent for measuring the substance to be measured in the sample according to (3) above.
(5) One or more substances selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof in measurement of a substance to be measured in a sample using a tetrazolium compound The method of suppressing the nonspecific color development of a tetrazolium compound by measuring in presence of.
(6) Measurement of a measurement target substance in a sample using a tetrazolium compound composed of one or more substances selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof Non-specific color development inhibitor of tetrazolium compounds.

  According to the present invention, in measurement of a measurement target substance in a sample using a tetrazolium compound, as a nonspecific color development inhibitor of the tetrazolium compound, Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5- One or more substances selected from benzenedisulfonic acid or salts thereof are present in or contained in the measurement reaction solution or measurement reagent, thereby affecting the effects of various interfering substances such as hemoglobin and ascorbic acid contained in the sample. It can suppress and can measure the density | concentration of the measuring object substance in a sample correctly.

(1) Tetrazolium compound In this invention, the measurement object substance in a sample is measured using a tetrazolium compound.
In the present invention, examples of the tetrazolium compound include tetrazolium blue [TB], nitrotetrazolium blue [nitro-TB], tetrazolium violet [TV], nitroblue tetrazolium [NBT], 3- (4,5-dimethyl-2- Thiazolyl) -2,5-diphenyl-2Htetrazolium [MTT], 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfenyl) -2H-tetrazolium salt [WST- 1], 2- (4-iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfenyl) -2H-tetrazolium salt [WST-3], 2- (2-methoxy- 4-Nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfenyl) -2H-tetra Rium salt [WST-8], 2,3,5-triphenyltetrazolium, 3- (4,5-dimethylthiazole-2-phenyl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfo Phenyl) -2H-tetrazolium salt [MTS] can be used.

  In the present invention, as the tetrazolium compound, a commercially available product can be used as it is. Further, the concentration of the tetrazolium compound is not particularly limited, but is preferably in the range of 3 μM to 30 mM, and particularly preferably in the range of 10 μM to 10 mM in the measurement reaction solution after mixing the biological sample and the measurement reagent.

(2) Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof In the measurement of a measurement target substance in a sample using the tetrazolium compound of the present invention, non-specificity of the tetrazolium compound One or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof as a selective color development inhibitor in a measurement reaction solution or a measurement reagent Measurement of the substance to be measured in the sample is performed by the presence or inclusion.
Here, examples of the Trinder reagent include phenol, a phenol derivative or a salt thereof, or an aniline derivative or a salt thereof.
Examples of phenol derivatives or salts thereof include known phenol derivatives such as 4-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, and salts thereof. Can be mentioned.
Examples of aniline derivatives or salts thereof include N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS). N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N- (3-sulfopropyl) aniline (HALPS), N-ethyl-N- (2- Hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2- Hydroxy-3-sulfopropyl) aniline (ALOS), N-ethyl-N- (3-sulfopropyl) aniline (ALPS), N-E Ru-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- Sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N- Sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-bis (4-sulfobutyl) -3,5-dimethylaniline (MADB), N, N-bis (4-sulfobutyl) -3-methylaniline (TODB), N- (2-cal Xylethyl) -N-ethyl-3-methylaniline (CEMB), N- (2-carboxyethyl) -N-ethyl-3,5-dimethoxyaniline (CEDB), N- (2-carboxyethyl) -N-ethyl -3-methoxyaniline (CEMO), N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxy Aniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl -N- (2-hydroxy-3-sulfopropyl) -m-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) anily N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-sulfopropylaniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline, or these Well-known things, such as a salt, can be mentioned.
In the present invention, as the Trinder reagent, a commercially available product can be used as it is. The concentration of the Trinder reagent is not particularly limited, but is preferably in the range of 0.01 to 5%, preferably in the range of 0.1 to 1% in the measurement reaction solution after mixing the sample and the measurement reagent. Is particularly preferred.

In the present invention, superoxide dismutase refers to an enzyme that catalyzes a reaction (disproportionation reaction) that generates oxygen molecules and hydrogen peroxide molecules from two molecules of superoxide anion (active oxygen).
In the present invention, any superoxide dismutase may be used, for example, those derived from animals such as humans or cows, those derived from plants such as soybeans, those derived from microorganisms such as bacteria or molds, etc. Can be mentioned.
This superoxide dismutase includes a superoxide dismutase prepared by a genetic recombination technique in which a superoxide dismutase gene such as a microorganism is incorporated into a microorganism such as Escherichia coli, or a microorganism whose properties have been improved by gene modification or the like. Dismutase and the like are also included. As the superoxide dismutase, a commercially available product can be used as it is.
The concentration of superoxide dismutase is not particularly limited, but it is preferably in the range of 0.05 to 10 units / mL in the measurement reaction solution after mixing the sample and the measurement reagent, A range of units / mL is particularly preferred.

In the present invention, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof can be a commercially available product as it is.
Further, the concentration of 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof is not particularly limited, but in the range of 0.01 to 5% in the measurement reaction solution after mixing the sample and the measurement reagent. The range of 0.1 to 1% is particularly preferable.

  The aforementioned Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof may be used alone, or two or more kinds may be used in combination. Moreover, when mixing and using, the density | concentration which combined them should just be said density | concentration range. Examples of the salt include alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as magnesium and calcium, ammonium salts, and hydrochlorides.

In addition, when the measurement method and the measurement reagent of the present invention are a one-step method (one reagent system), a Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid to be included in the measurement reagent Alternatively, the concentration of the salt may be in the above range, and in the case of a multiple step method (multiple reagent system) of two step method (two reagent system) or more, When the mixture is mixed at a ratio of the addition amount of the above, the concentration of the Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof in the measurement reaction liquid after mixing is within the above range. What is necessary is just to determine the density | concentration in a measuring reagent so that it may become.
For example, in the case of the two-step method (two-reagent system), the concentration of the Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof in the measurement reaction liquid after mixing is the above concentration. To be within the range, Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof may be included in either the first reagent or the second reagent.
In addition, if the concentration of the Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof in the measurement reaction liquid after mixing falls within the above concentration range, the Trinder reagent, Super Oxido dismutase or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof may be included in both the first reagent and the second reagent.

(3) Other components in measurement In the present invention, in addition to the above-described components, electron acceptors such as dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, electron carriers, etc. Components used in the measurement reaction, known preservatives, stabilizers, and the like can be appropriately used as necessary.
The dehydrogenase used in the present invention is an enzyme that catalyzes a reaction that causes an oxidation reaction between an electron acceptor and a substance to be measured or a substance produced from the substance to be measured to produce a reduced form of the electron acceptor. That means. For example, known dehydrogenases such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase, 3α-hydroxysteroid dehydrogenase, lactate dehydrogenase, L-glutamate dehydrogenase or leucine dehydrogenase are listed. be able to.
Examples of the electron carrier used in the present invention include diaphorase, phenazine methosulfate (PMS), 1-methoxyphenazinium methyl sulfate (1-methoxy PMS), and 9-dimethylaminobenzo-α-. Mention may be made of substances such as phenazoxonium chloride (Meldra Blue). As these electron carriers, commercially available products can be used as they are.

(4) pH at the time of measurement
In the present invention, the pH range at the time of measurement of a substance to be measured in a sample may be appropriately set depending on the optimum pH and stability of an enzyme or the like that is normally used in measurement using a tetrazolium compound.
Moreover, as a buffer solution used so that it may become said pH range, the conventionally well-known buffer solution which has a buffer capacity in the said pH range can be used suitably.
Examples of buffers that can be used include phosphoric acid, tris (hydroxymethyl) aminomethane, glycylglycine, MES, Bis-Tris, ADA, ACES, Bis-Tris propane, PIPES, MOPSO, MOPS, Examples of the buffer solution include BES, HEPES, TES, DIPSO, TAPSO, POPSO, HEPPS, HEPPSO, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS, and salts thereof.

(5) Composition of reagents, etc., and concentration of constituents, etc. The measurement method and measurement reagent of the present invention may be implemented and configured by a one-step method (one reagent system), or two steps (two reagents) or more. You may implement and comprise by the multistep method (multiple reagent system).
When the measurement method and the measurement reagent of the present invention are a one-step method (one-reagent system), the concentration, pH, and the like of each component described above may be in the above ranges, and the multiple-step method ( In the case of a multi-reagent system), when mixing at the ratio of the respective addition amounts when measuring the substance to be measured, the concentration and pH of the constituent components in the measurement reaction liquid after the mixing are What is necessary is just to determine the density | concentration of the component of each reagent, etc. so that it may become the concentration range of a component, pH range, etc.

(6) Sample In the present invention, the sample is intended to measure the substance to be measured in the sample, and is not particularly limited as long as it is such.
Examples of such samples include human or animal blood, serum, plasma, urine, stool, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, brain and other organs, hair, skin, nails and muscles. Or the extract of tissues, such as a nerve, and a cell etc. are mentioned.

(7) Substance to be measured In the present invention, the substance to be measured is a substance that can use a dehydrogenase for the substance to be measured in the presence of an electron acceptor, or a substance generated from the substance to be measured. On the other hand, there is no particular limitation as long as the dehydrogenase can be used in the presence of an electron acceptor. For example, bile acid, glucose, 1,5-anhydroglucitol, fructose, triglyceride, phospholipid , Total cholesterol, HDL cholesterol, LDL cholesterol, free fatty acid, urea nitrogen, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, creatinine and the like.

(8) Measuring method or measuring reagent of substance to be measured The present invention comprises one or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof. The method or reagent for measuring a substance to be measured in a sample using a tetrazolium compound, which is present in a measurement reaction solution or a reagent as a nonspecific color development inhibitor of a tetrazolium compound. Therefore, the basic measurement method for the substance to be measured is based on a known measurement method in which an electron acceptor and a dehydrogenase are allowed to act on a sample, and the reduced form of the electron acceptor is measured using a tetrazolium compound. Can be done.
When measuring a substance to be measured in a sample, for example, the sample and one or more substances selected from a Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof And a reagent containing The mixture of the sample and the reagent is mixed with a reagent containing an electron acceptor, a dehydrogenase, and a tetrazolium compound, an enzyme reaction is performed, and a reductant of the generated electron acceptor and a tetrazolium compound are colored. The concentration of the substance to be measured in the biological sample can be measured by measuring the absorbance of the reaction solution before and after the color development and determining the amount of change in absorbance due to the tetrazolium compound.
The measurement can be carried out by either rate assay method or endpoint method.

(9) Method for inhibiting non-specific color development of tetrazolium compound The method for inhibiting non-specific color development of a tetrazolium compound in the present invention is a method for measuring a substance to be measured in a sample by using a tender reagent, superoxide dismutase, or 1 , 2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof is measured in the presence of one or more substances.
In measurement of the substance to be measured in this sample, measurement is performed in the presence of one or more substances selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof. By doing so, even when interfering substances such as ascorbic acid and hemoglobin are mixed in the sample, it is possible to suppress the occurrence of errors in measured values due to the interfering substances, and to measure the measurement target substance with high accuracy. A value can be obtained.
In addition, the components, samples, conditions, and the like of the reagents used in the method for suppressing nonspecific color development of the tetrazolium compound in the present invention are as described above.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

(Confirmation of non-specific color development inhibitory effect of tetrazolium compound according to the present invention-1)
The inhibitory effect of non-specific color development was confirmed when Trinder reagent, superoxide dismutase, and 1,2-dihydroxy-3,5-benzenedisulfonic acid were contained in a measurement reagent using a tetrazolium compound.

1. Preparation of measuring reagent (1) Preparation of first reagent for bile acid measurement 18 kinds of Trinder reagent, superoxide dismutase, or 1, 2, 1, 2, and 2 shown in Table 1 so that the following reagent components have respective concentrations described -Dihydroxy-3,5-benzenedisulfonic acid was dissolved in pure water so as to have the concentrations shown in Table 1, and the pH was adjusted to 7.2 (20 ° C). Twenty kinds of first reagents for measuring bile acids each containing (or not containing) oxiddismutase or 1,2-dihydroxy-3,5-benzenedisulfonic acid were prepared.
PIPES 100 mM
Pyruvate 10 mM
Ascorbate oxidase 0.5 unit / mL

(2) Preparation of Second Reagent for Bile Acid Measurement The following reagent components are dissolved in pure water so as to have the respective concentrations described above, pH is adjusted to 3.0 (20 ° C.), and second bile acid measurement second reagent is prepared. Reagents were prepared. (In order to measure only non-specific color development of the tetrazolium compound, 3α-hydroxysteroid dehydrogenase, which is an enzyme necessary for bile acid measurement, was not included.)
Glycine 10 mM
EDTA 1 mM
Nicotinamide adenine dinucleotide 3mM
WST-1 300μM
1-methoxy-5-methylphenazinium methyl sulfate 15 μM

2. Sample Preparation A sample prepared by adding hemoglobin to human serum so that the addition concentration was 250 or 500 mg / dL.
Further, a sample to which physiological saline was added instead of hemoglobin was used as a hemoglobin-free sample (hemoglobin added concentration was 0 mg / dL).

3. Measurement of Sample For each of the two samples, measurement is performed using the 20 types of the first reagent for bile acid measurement and the second reagent for bile acid measurement prepared in 1 above, and the measurement value is obtained by non-specific color development of the tetrazolium compound. The error was confirmed.
The measurement with the measurement reagent of the present invention is carried out with a 7170S type automatic analyzer manufactured by Hitachi, Ltd., and 160 μL of the first reagent for bile acid measurement prepared in 1 above is added to each 8 μL of the sample prepared in 2 above. After mixing, the mixture was reacted at 37 ° C. for 5 minutes, and then 80 μL of the second reagent for bile acid measurement prepared in 1 was added and reacted at 37 ° C. for 5 minutes. Absorbance at a main wavelength of 450 nm and a subwavelength of 700 nm immediately before and after the addition of the second reagent for bile acid measurement was measured, and the difference was obtained. Then, the sample (standard solution) with a known bile acid concentration is measured as described above, and the measured value (absorbance difference) of the standard solution is compared with the measured values (absorbance difference) of the three types of samples. The values obtained by converting the measured values (absorbance difference) of the three types of samples into bile acid concentrations were obtained.

4). Measurement results Table 1 shows the measurement results of the samples.

In this example, since the measurement reagent does not contain 3α-hydroxysteroid dehydrogenase, which is a dehydrogenase necessary for bile acid measurement, the measured value of each sample should be originally “0”. is there. That is, all measured values obtained by measuring each sample are positive errors caused by non-specific color development of the tetrazolium compound.
As apparent from Table 1, when the first reagent does not contain Trinder reagent, superoxide dismutase or 1,2-dihydroxy-3,5-benzenedisulfonic acid (no addition), the influence of hemoglobin in the sample As a result, it can be seen that there is a positive error in the measured value of each sample that should be “0”. It can also be seen that as the concentration of added hemoglobin in the sample increases, the measured value of each sample increases and the degree of positive error increases.
On the other hand, when the first reagent contains Trinder reagent, superoxide dismutase or 1,2-dihydroxy-3,5-benzenedisulfonic acid, the positive error of this measured value is reduced and improved. I understand.

  From these, in the measurement of the measurement target substance in the sample using the tetrazolium compound, by performing the measurement in the presence of a Trinder reagent, superoxide dismutase or 1,2-dihydroxy-3,5-benzenedisulfonic acid, Even when hemoglobin is mixed in the sample, it is possible to suppress the occurrence of errors in the measured value due to hemoglobin, and as a result, non-specific color development of the tetrazolium compound due to hemoglobin, which is a disturbing substance in the sample It became possible to suppress and it was confirmed that an accurate measured value can be obtained.

(Confirmation of non-specific color development inhibitory effect of tetrazolium compound according to the present invention-2)
The inhibitory effect of non-specific color development was confirmed when the Trinder reagent was included in a measurement reagent using a tetrazolium compound.

1. Preparation of measurement reagent (1) Preparation of first reagent for bile acid measurement The following reagent components are dissolved in pure water so as to have the respective concentrations described above, pH is adjusted to 7.2 (20 ° C.), and Trinder reagent is used. Five types of first reagents for measuring bile acids with different HDAOS concentrations were prepared.
HDAOS 0%, 0.01%, 0.1%, 0.5% or 1%
PIPES 100 mM
Pyruvate 10 mM
Ascorbate oxidase 0.5 unit / mL

(2) Preparation of second reagent for bile acid measurement The second reagent for bile acid measurement prepared in (1) of Example 1 was used as it was.

2. Preparation of sample A sample prepared by adding ascorbic acid to human serum so that the addition concentration was 10, 20, 30, 40, or 50 mg / dL.
Moreover, what added the physiological saline instead of ascorbic acid was made into the sample without an ascorbic acid (ascorbic acid addition density | concentration is 0 mg / dL).

3. Measurement of Sample For each sample of 2 above, the five kinds of first reagent for bile acid measurement and the second reagent for bile acid measurement prepared in 1 were measured in the same manner as 3 in Example 1, and a tetrazolium compound An error in the measured value due to non-specific color development was confirmed.

4). Measurement results Table 2 shows the measurement results of the samples. In addition, in order to clarify the influence of ascorbic acid, the bile acid equivalent value in Table 2 is the measured value of each ascorbic acid-added sample, the measured value of the ascorbic acid-free sample (ascorbic acid added concentration is 0 mg / dL). It is shown as a difference with respect to.

In this example, since the measurement reagent does not contain 3α-hydroxysteroid dehydrogenase, which is a dehydrogenase necessary for bile acid measurement, the measured value of each sample should be originally “0”. is there. That is, all measured values obtained by measuring each sample are positive errors caused by non-specific color development of the tetrazolium compound.
As apparent from Table 2, when HDAOS is not included in the first reagent, a positive error occurs in the measured value of each sample that should be “0” due to the influence of ascorbic acid in the sample. I understand that. Furthermore, it can be seen that as the concentration of added ascorbic acid in the sample increases, the measured value of each sample increases and the degree of positive error increases.
In Example 1, the first reagent contains ascorbic acid oxidase for suppressing the influence of ascorbic acid, but when the first reagent does not contain HDAOS, the measured value of bile acid It can be seen that a positive error has occurred. That is, it can be seen that ascorbate oxidase does not suppress the nonspecific color development of the tetrazolium compound.
On the other hand, when HDAOS is contained in the first reagent, it can be seen that the positive error of the measured value is reduced and improved.

  From these facts, in the method for measuring a substance to be measured in a sample using a tetrazolium compound, even if ascorbic acid is mixed in the sample by performing measurement in the presence of a Trinder reagent, this It is possible to suppress the occurrence of errors in measurement values due to ascorbic acid, and as a result, it is possible to suppress nonspecific color development of tetrazolium compounds due to ascorbic acid, which is an interfering substance in the sample, and to obtain accurate measurement values It was confirmed that it was possible.

(Confirmation of non-specific color development inhibitory effect of tetrazolium compound according to the present invention-3)
The inhibitory effect of non-specific color development was confirmed when the Trinder reagent was included in a measurement reagent using a tetrazolium compound.

1. Preparation of measurement reagent (1) Preparation of first reagent for bile acid measurement The following reagent components are dissolved in pure water so as to have the respective concentrations described above, pH is adjusted to 7.2 (20 ° C.), and Trinder reagent is used. Five types of first reagents for measuring bile acids with different HDAOS concentrations were prepared.
HDAOS 0%, 0.01%, 0.05%, 0.1% or 0.2%
PIPES 100 mM
Pyruvate 10 mM
Ascorbate oxidase 0.5 unit / mL

(2) Preparation of second reagent for bile acid measurement The second reagent for bile acid measurement prepared in (1) of Example 1 was used as it was.

2. Sample Preparation Forty kinds of human serum samples (sample numbers: 1 to 40) were prepared.

3. Measurement of Sample For each sample of 2 above, the five kinds of first reagent for bile acid measurement and the second reagent for bile acid measurement prepared in 1 were measured in the same manner as 3 in Example 1, and a tetrazolium compound An error in the measured value due to non-specific color development was confirmed.

4). Measurement results Table 3 shows the measurement results of the samples.

In this example, since the measurement reagent does not contain 3α-hydroxysteroid dehydrogenase, which is a dehydrogenase necessary for bile acid measurement, the measured value of each sample should be originally “0”. is there. That is, all measured values obtained by measuring each sample are positive errors caused by non-specific color development of the tetrazolium compound.
As is clear from Table 3, when HDAOS is not contained in the reagent, a positive error occurs in the measured value of each sample that should be “0” due to the influence of some interfering substances in the sample. I understand that.
On the other hand, when HDAOS is contained in the first reagent, it can be seen that the positive error of the measured value is reduced and improved.

  From these facts, in the measurement of the measurement target substance in the sample using the tetrazolium compound, this interference can be obtained even if the interfering substance is mixed in the sample by performing the measurement in the presence of the Trinder reagent. It is possible to suppress the occurrence of errors in measured values due to substances, and as a result, it is possible to suppress nonspecific color development of tetrazolium compounds due to interfering substances in the sample, and it is confirmed that accurate measured values can be obtained. It was.

Claims (6)

  In the method for measuring a substance to be measured in a sample using a tetrazolium compound, one or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof, A method for measuring a substance to be measured in a sample, which is present in a measurement reaction solution.   One or more substances selected from a Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof are present as a nonspecific color development inhibitor of a tetrazolium compound. 1. A method for measuring a substance to be measured in a sample according to 1.   A measuring reagent for a substance to be measured in a sample using a tetrazolium compound contains one or more substances selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof. A reagent for measuring a substance to be measured in a sample.   One or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof are contained as a non-specific color development inhibitor of a tetrazolium compound. 3. A reagent for measuring a substance to be measured in the sample according to 3.   In the measurement of a substance to be measured in a sample using a tetrazolium compound, in the presence of one or more substances selected from Trinder reagent, superoxide dismutase, 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof A method for suppressing non-specific color development of a tetrazolium compound by performing measurement in   Tetrazolium at the time of measurement of a substance to be measured in a sample using a tetrazolium compound, comprising one or more substances selected from Trinder reagent, superoxide dismutase, or 1,2-dihydroxy-3,5-benzenedisulfonic acid or a salt thereof A non-specific color development inhibitor for compounds.