KR20180004027A - Analytical kit for enzyme activity - Google Patents

Abstract

The present invention relates to a kit for analyzing enzymatic activities. More specifically, the present invention relates to a method for producing an analysis kit for detection of risk factors causing hemolytic anemia due to deficiency in activities of glucose-6-phsphate dehydrogenase (G6PD). By screening deficiency in activities of G6PD, detection of activities at various levels is possible. In addition, it is also possible to determine the level of activities of G6PD with ease by creating data on individual samples having such features through observation with naked eyes or an image quantitative analysis.

Description

[0001] The present invention relates to a kit for analyzing an enzyme activity,

The present invention relates to an enzyme activity assay kit and an enzyme activity assay method, and more particularly, to an assay kit and an assay kit for rapidly and accurately screening a specific enzyme activity in blood using a human blood sample, ≪ / RTI >

Glucose-6-phsphate dehydrogenase (G6PD) is an important enzyme that converts carbohydrates into energy and protects red blood cells from side effects that may occur during infection or drug treatment.

G6PD deficiency is the most common enzyme deficiency disease and is inherited as X-chromosomal recessive disorder, and it is known that there is a difference in severity according to race and sex. In normal condition, only 3% of the enzyme is used. Therefore, most patients do not have symptoms. However, when oxidative stress is caused by infection, medicine, etc., NADPH production disorder accumulates intracellular oxidants and causes hemolysis.

G6PD acts on the hexose monophosphate (HMP) pathway as glucose-6-phosphate is converted to 6-phosphate gluconate, and coenzyme NADP is reduced to NADPH. When G6PD is deficient, NADPH can not be produced. Glutathione Glutathione (GSH) metabolism can not start.

GSH in red blood cells can only be converted to GSH reduced by NADPH to neutralize the oxidizing agent produced in red blood cells by infection or certain drugs. In G6PD-deficient red blood cells, oxidants such as over ^- oxygen (O ^2- ) and hydrogen peroxide accumulate, which causes the hemoglobin to oxidize to become methemoglobin, and further progresses to sulfhemoglobin, which precipitates and coalesces into Heinz Heinz 'bodies, which are attached to the red blood cell membrane, causing hemolysis.

Unlike other cells in the body, red blood cells are the metabolic pathway that produces NADPH, and the pathway using G6PD is unique. It is relatively vulnerable to oxidative stress as compared with other cells. When exposed to oxidative stress, red blood cell hemolytic anemia appear.

Because the enzyme activity is transmitted through the X-chromosome, the activity is reduced to less than 5% in men and in homozygous females, and various levels of enzyme activity are reported in women with heterozygous females. G6PD deficiency is the most common cause of hemolysis and jaundice in neonates in erythrocyte enzyme deficiency. When a newborn with G6PD deficiency develops acidosis due to infection, hypoxia, dehydration, or exposure to drugs, hemolysis occurs and transfusion is required do. The major factors that cause severe G6PD deficiency symptoms include overexposure to chemicals such as anti-malarials, aspirin, phenacetin, sulfa, naphthalene, and bacterial or viral infections.

However, when primaquine, pamaquine, chloroquine, etc. are used as a malaria treatment for G6PD genetic mutation, the accumulation of oxidizing substances in erythrocytes accelerates the side effects caused by erythrocyte disruption It is often the case that it is induced and, in severe cases, it dies. Therefore, there is a need to develop an easy way to obtain information on the types of therapeutic agents available.

Korean Patent No. 10-1365721 (Apr. 21, 2014) discloses an apparatus for diagnosing a mutation type of G6PD gene, and Korean Patent No. 10-1751205 (Jun. 2017) Discloses a lateral flow chromatography analysis method using a strip for analysis and a test apparatus including the same.

Therefore, the present inventors developed an assay kit capable of quickly and accurately screening the activity of specific enzymes in blood and visually reading the activity of the infection or enzyme. As a result, a kit that can confirm the activity with a clear color change by comparing the colors of the test pad and the control pad was manufactured. Through this, a glucose-6-phsphate dehydrogenase (G6PD) And can be usefully used as an assay kit for identifying risk factors capable of causing hemolytic anemia.

An object of the present invention is to provide a kit for analyzing an enzyme activity.

Another object of the present invention is to provide a method for analyzing enzyme activity.

The present invention relates to a kit for analyzing enzyme activity, and includes a kit for assaying an enzyme activity, which can accurately screen specific enzyme activity in blood and visually read it.

Hereinafter, the present invention will be described in more detail.

One aspect of the invention is a dye compound having specificity for an analyte; A test pad on which a reactant in which a sample is reacted with the dye compound is developed; And a control pad on the downstream side of the test pad, wherein the control pad is pretreated with a dye reducing agent.

The basic principle of the present invention is to convert the formazan dye sample using the activity of glucose-6-phsphate dehydrogenase (G6PD), and the result of enzyme reaction is cellulose, glass fiber And a polyester in which a capillary phenomenon is induced.

The kit may further include a sample pad for injecting a sample on the upstream side of the test pad.

The dye compound may be pre-treated on a sample pad, pretreated on a separate pad, lyophilized, and then placed as a dye pad between the sample pad and the test pad. However, the present invention is not limited thereto.

The sample pad may be selected from the group consisting of glass fiber, cellulose and polyester, and may be, for example, glass fiber, but is not limited thereto.

The dye pad may be selected from the group consisting of cellulose, glass fiber and polyester, and may be, for example, cellulose, but is not limited thereto.

In an embodiment of the present invention, when the dye compound is pretreated on the sample pad without the dye pad, the sample pad may further include a buffer, a surfactant, or a mixture thereof as a substance to be pretreated.

The buffer was washed with 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffered saline (PBS) buffer And / or a Tris buffer.

The surfactant may utilize Triton X-100 and / or Tween 20, but is not limited thereto.

The inspection pad can be confirmed through an inspection area which can be confirmed after the plastic housing is assembled, and for example, the analysis result can be confirmed by a change in color identified in the pad, , But is not limited thereto.

The test pad may be, but is not limited to, cellulose. When cellulose is used as a test pad, the development speed of the sample is excellent and the color of the dye is shown to be deep.

The kit may further include a control pad on the downstream side of the test pad for determining whether the color of the test pad changes.

The check pad is a check object to check the analysis result, and it can be confirmed whether the check is valid or not. The verification pad can be confirmed through a check area which can be confirmed from the appearance after assembling the plastic housing. For example, The degree of change can be confirmed by contrasting with the color of the control pad, but is not limited thereto.

The control pad may be selected from the group consisting of cellulose, glass fiber and polyester, and may be, for example, cellulose, but is not limited thereto.

The kit may additionally include an absorbent pad on the downstream side of the control pad.

The absorbent pad may be, but is not limited to, cellulose.

As used herein, the term "analyte" refers to a biological material, biological sample, or specific component in a sample that has a specific activity that is the subject of analysis to identify one of the results, such as presence, absence, or concentration.

The analyte may be at least one selected from the group consisting of proteins such as antigens, antibodies, enzymes, carbohydrates, lipids, nucleic acids and metabolites, and may be, for example, enzymes, but is not limited thereto.

These enzymes include glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine-beta-hydroxylase, glutathione synthesis (AST), an alanine aminotransferase (ALT), an alkaline phosphatase (ALP), and a gamma-glutamyltransferase (gamma-glutamyltransferase), as well as enzymes such as glutathione synthetase, aspartate aminotransferase -glutamyl transferase (GGT), and may be, for example, G6PD, but is not limited thereto.

The analyte may also be a toxic chemical, an environmental hormone or a neurotransmitter.

As used herein, the term "toxic chemical" means a harmful chemical having acute toxicity, fouling toxicity, mutagenicity and carcinogenicity, and includes cadmium, cadmium compounds, chlorine, hydrogen chloride, fluorine, silicon fluoride, lead, Nitrogen oxides, and nitrogen oxides.

The term " environmental hormone " in this specification means an industrial substance corresponding to an exogenous endocrine disrupting chemical substance and may be one or more selected from the group consisting of insecticides, pesticides, organic heavy metals, dioxins and synthetic estrogens used as pharmaceuticals have.

As used herein, the term "neurotransmitter" refers to a substance that is secreted in a neuron, which acts to increase or decrease the potential of adjacent neurons.

The analyte may be, but is not limited to, one which is present in the body fluid of the specimen or is separated from the body fluids of the specimen.

The specimen may be an animal, such as a mammal, rodent, reptile, or bird, and may be, for example, a mammal, but is not limited thereto.

As used herein, the term "dye compound" refers to a substance that exhibits color so that the analysis result can be visually confirmed according to the specific activity with the analyte to be reacted.

The dye compound may be selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2-methoxy- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carbanilide (XTT) Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium, tetrazolium chloride ( tetrazolium chloride) and 2,3,5-triphenyl-2H-tetra Tetrazolium chloride (TTC), and may be, for example, 2,6-dichlorophenolindophenol, but it is not limited thereto. no.

According to one embodiment of the present invention, when the blood lacking G6PD is used as a sample, DCPIP is not reduced when it reacts with DCPIP, which is a dye compound, so that the blue color is maintained. As a result, hemoglobin and blue dye are mixed Cyan or brown.

The kit may further comprise nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent that reduces NADP to NADPH.

The NADP derivative may be selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate, β-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, nicotinamide 1, N6- ethenoadenine dinucleotide and nicotinamide 1, and N6-ethenoadenine dinucleotide phosphate. However, the present invention is not limited thereto.

As used herein, the term "electron transfer agent" refers to a substance that contributes to the NADP + -reduced response to NADPH.

The electron transfer agent may be 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase, Meldola Blue, and may be, for example, PMS, but is not limited thereto.

The term "dye reducing agent" in this specification means a substance that reduces the dye compound.

The dye reducing agent may be ascorbic acid, but is not limited thereto.

According to one embodiment of the present invention, the reactant reacted with the dye compound DCPIP to analyze the sample is developed on the test pad and the control pad, and DCPIP contained in the reactant by the pretreated ascorbic acid in the control pad is blue The color of the test pad is compared with the color of the control pad to confirm the result of the analysis.

Another aspect of the present invention is an enzyme activity assay method comprising the steps of:

A sample injection step of injecting a sample into an analytical reagent containing a dye compound having specificity for an analyte; And

A color matching step of comparing the color change appearing in the reagent of the analysis reagent and the sample with the control pad.

The color matching step may be performed after the reactant is developed into the test pad and the control pad.

The control pad may be located on the downstream side of the inspection pad, and the dye reducing agent may be pretreated.

The control pad reduces the color of the dye by the pretreated dye reducing agent, so that the hue appearing on the test pad changes colorless on the control pad, and the color change on the test pad can be clearly determined.

The analyte may be at least one selected from the group consisting of proteins such as antigens, antibodies, enzymes, carbohydrates, lipids, nucleic acids and metabolites, and may be, for example, enzymes, but is not limited thereto.

In addition, the analyte may be present in the sample body fluid or separated from the sample body fluid, but is not limited thereto.

The specimen may be an animal, such as a mammal, rodent, reptile, or bird, and may be, for example, a mammal, but is not limited thereto.

These enzymes include glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine-beta-hydroxylase, glutathione synthesis (AST), an alanine aminotransferase (ALT), an alkaline phosphatase (ALP), and a gamma-glutamyltransferase (gamma-glutamyltransferase), as well as enzymes such as glutathione synthetase, aspartate aminotransferase -glutamyl transferase (GGT), and may be, for example, G6PD, but is not limited thereto.

The dye compound may be selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (INT), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3- -5-sulfolenyl) -2H-tetrazolium-5-carbanilide (XTT), nitroblue tetrazolium (NBT), 3- (4,5-dimethylthiazol- (MTS), tetrazolium, tetrazolium chloride, and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) And may be, for example, 2,6-dichlorophenolindophenol, but is not limited thereto.

The analytical reagent may further include nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent that reduces NADP to NADPH.

The NADP derivative may be selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate phosphate, beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, nicotinamide 1, N6- ethenoadenine dinucleotide and nicotinamide 1, and N6-ethenoadenine dinucleotide phosphate. However, the present invention is not limited thereto.

The electron transfer agent may be 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase, Meldola Blue, and may be, for example, PMS, but is not limited thereto.

(2, 6-dichlorophenolindophenol; DCPIP), nicotinamide adenine dinucleotide phosphate (NADP), NADP derivatives and glucose-6-phosphate (G6P) In the case of using two or more analytical reagents selected from the group, the analytical mixture may be absorbed on the solid support or solid support, dried, and then dispensed in a constant amount.

The solid support may be, for example, a pad, but is not limited thereto.

The dye reducing agent may be ascorbic acid, but is not limited thereto.

Since the enzyme activity assay method of the present invention relates to a kit for assaying enzyme activity, which is another aspect of the present invention, duplicate contents are omitted in order to avoid the excessive complexity described in the present specification.

The enzyme activity assay kit of the present invention can be used to accurately identify a specific enzyme activity in the blood and visually read it, and thus to effectively identify a risk factor for causing hemolytic anemia due to G6PD activity deficiency.

FIG. 1A is a schematic diagram showing the structure of a kit for analyzing hemolytic anemia-related enzyme activity. FIG.
1B is a photograph of an enzyme activity assay kit prepared according to an embodiment of the present invention.
FIG. 2 is a photograph showing that the color change of the test pad can be confirmed by contrasting with the control pad by injecting samples different in enzyme activity into the enzyme activity assay kit of the present invention.
FIG. 3 is a photograph showing blood levels of G6PD activity deficiency in different levels. FIG.
FIG. 4 is a photograph comparing the easiness of judging the result of the analysis of the enzyme activity assay kit (right upper side) of the present invention with a kit for assaying the activity of an enzyme which has been commercialized.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Preparation of reaction solution

Sample pad Pretreatment solution (1 x PBS 32 mL, 461 uL of D-glucose 6 phosphate disodium salt hydrate, 250 mg / mL G6P), 50 mg / mL NADP (b-Nicotinamide adenine dinucleotide phosphate hydrate, Sigma, N5755) 192 uL, 480 uL of 100 mg / mL maleimide (Sigma, 129585), 1 mg of methoxy-5-methylphenazium methyl sulfate (hereinafter referred to as PMS, Sigma M8640) uL), 2,6-dichlorophenolindophenol (DCPIP), which changes from blue to colorless as NADP + is reduced to NADPH by action from red blood cells, 4- (2-hydro- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (hereinafter referred to as HEPES) buffer and a surfactant were prepared.

Example 2. Fabrication of assay kit

As shown in FIG. 1A, a sample pad, a dye pad, a test pad, a control pad, and an absorption pad are arranged on the support plate from the upstream side to the downstream side, Respectively.

The sample pad is made of glass fiber material and corresponds to 1.92 mg per sheet. 1 ml of PBS, 461 μL of 250 mg / mL G6P (D-glucose 6 phosphate disodium salt hydrate, Sigma G7250), 192 μL of 50 mg / mL NADP (b-Nicotinamide adenine dinucleotide phosphate hydrate, Sigma, N5755) 480 μL of maleimide (Sigma, 129585) and 96 μL of 20 mg / mL 1-methoxy-5-methylphenazinium methyl sulfate (PMS, Sigma M8640) Mixed and infiltrated, and lyophilized.

The dye pad was infiltrated with 10 ug / ml of 2,6-dichlorophenolindophenol (DCPIP) in the cellulose (Ahlstrom, ap 31) pad and lyophilized because DCPIP was sensitive to high temperature.

The test pads were prepared with a cellulose pad and were not pretreated separately.

A control pad was prepared by infiltrating cellulose pad with 1 M ascorbic acid (Sigma, 255564), followed by hot-air drying at 35 ° C for 2 hours, and cutting to a length of 10 mm.

The absorbent pad was prepared and attached to a cellulose pad.

As shown in FIG. 1B, the assay kit of the present invention was completed by inserting and supporting the support plate into a plastic housing.

Example 3. Visual analysis of risk factors for hemolytic anemia

150 μL of 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer was prepared, and 5 μL of normal blood or G6PD deficiency 5 μl of blood was mixed to prepare a normal sample and a G6PD deficient sample, which were then examined in an assay kit.

As can be seen from FIG. 2, the enzyme activity assay kit of the present invention can be analyzed in comparison with the color of the control pad because the degree of change of the color of the test pad increases according to the activity of the enzyme.

In the sample (normal blood) with an enzyme activity of 100%, both the test region (test pad) and the control region (control pad) were red with the same blood. The normal blood contains G6PD, so pink is produced by the reduction of the DCPIP dye, resulting in a red color mixed with hemoglobin.

However, when a sample with an enzyme activity of 10% (blood of a person who is at risk of hemolytic anemia due to G6PD deficiency) is reacted, the DCPIP dye is not reduced to maintain the blue color, and hemoglobin and blue dye are mixed to form cyan . As a result, it was confirmed that the test region appeared green and the control region was red, so that it could be distinguished from normal blood clearly.

Figure 3 shows the difference in color change due to differences in the degree of G6PD deficiency. Depending on the degree of deficiency, it can be seen that not only green but also brown can appear.

As can be seen from FIG. 4, the enzyme activity assay kit of the present invention (upper right hand side) clearly shows that the assay results are clearly distinguished from other commercially available enzymatic activity assay kits.

Claims (16)

A dye compound having specificity for an analyte;
A test pad on which a reactant in which a sample is reacted with the dye compound is developed; And
A control pad on the downstream side of the test pad, wherein the control pad is pretreated with the dye reducing agent;
A kit for assaying an enzyme activity. The kit for analyzing enzyme activity according to claim 1, wherein the analyte is at least one selected from the group consisting of proteins such as antigens, antibodies, enzymes, carbohydrates, lipids, nucleic acids and metabolites. The method of claim 1, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine- beta-hydroxylase, glutathione synthetase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma- Wherein the enzyme is at least one selected from the group consisting of gamma-glutamyl transferase (GGT). The method according to claim 1, wherein the dye compound is selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2- Methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5 (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium- -carbanilide (XTT), Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium ), Tetrazolium chloride and 2,3,5- Lee phenyl -2H- tetrazolium chloride (2,3,5-triphenyl-2H- tetrazolium chloride; TTC) which is at least one member selected from the group consisting of enzyme activity analysis kit. The kit for analyzing enzyme activity according to claim 1, wherein the kit further comprises an electron transfer agent for reducing nicotinamide adenine dinucleotide phosphate (NADP), NADP derivative or NADP to NADPH. 6. The method of claim 5, wherein the NADP derivative is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide wherein the kit is one or more selected from the group consisting of nicotinamide 1, N6-ethenoadenine dinucleotide and nicotinamide 1, and N6-ethenoadenine dinucleotide phosphate. 6. The method of claim 5, wherein the electron transfer agent is selected from the group consisting of 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase Diaphorase, and Meldola Blue. ≪ Desc / Clms Page number 19 > The kit for analyzing enzyme activity according to claim 1, wherein the dye reducing agent is ascorbic acid. An enzyme activity assay method comprising the steps of:
A sample injection step of injecting a sample into an analytical reagent containing a dye compound having specificity for an analyte; And
A color matching step of comparing the color change appearing in the reagent of the analysis reagent and the sample with the control pad. 10. The method according to claim 9, wherein the analyte is at least one selected from the group consisting of proteins such as antigens, antibodies, enzymes, carbohydrates, lipids, nucleic acids and metabolites. 10. The method of claim 9, wherein the enzyme is selected from the group consisting of glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine- beta-hydroxylase, glutathione synthetase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma- Wherein the enzyme activity is at least one selected from the group consisting of gamma-glutamyl transferase (GGT). The method of claim 9, wherein the dye compound is selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) (INT), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3- -5-sulfolenyl) -2H-tetrazolium-5-carbanilide (XTT), nitroblue tetrazolium (NBT), 3- (4,5-dimethylthiazol- (MTS), tetrazolium, tetrazolium chloride, and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) Wherein the enzyme activity is at least one selected from the group consisting of: 10. The method according to claim 9, wherein the analytical reagent further comprises a nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent for reducing NADP to NADPH. 14. The method of claim 13, wherein the NADP derivative is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide wherein the enzyme activity is at least one selected from the group consisting of nicotinamide 1, N6-ethenoadenine dinucleotide and nicotinamide 1, and N6-ethhenoadenine dinucleotide phosphate. 14. The method of claim 13, wherein the electron transfer agent is selected from the group consisting of 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase Diaphorase, and Meldola Blue. ≪ Desc / Clms Page number 20 > 10. The method according to claim 9, wherein the dye reducing agent is ascorbic acid.

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