Method for quantifying ethanolamine phosphate, oxidoreductase for quantification, composition for quantification, kit for quantification, sensor chip, and sensor

Abstract

Provided are: a method for quantifying ethanolamine phosphate, whereby a minute quantity of ethanolamine phosphate can be measured; and an oxide reductase for quantification, a quantification composition, a quantification kit, a sensor chip, and a sensor that are used in said quantification method. According to an embodiment of the present invention, there is provided a method for quantifying ethanolamine phosphate, the method including causing a phosphatase to act on a sample to convert ethanolamine phosphate included in the sample to ethanolamine, and causing an oxide reductase to act on the ethanolamine.

Description

Technical Field

[0001] The present invention relates to a method for quantifying ethanolamine phosphate, an oxidoreductase for quantification, a composition for quantification, a kit for quantification, a sensor chip, and a sensor.

Background Art

[0002] Ethanolamine phosphate (EAP) contained in human blood is known to be a biomarker for diagnosing depression (Patent Document 1). Further, Non-Patent Document 1 reports that it is possible to diagnose depression using a reference value of the concentration of EAP in plasma of 1.5 μM or less.

[0003] However, for the measurement method of EAP, a complicated measurement method such as capillary electrophoresis time-of-flight mass spectrometry has been used. As a simpler measurement method, Patent Document 2 describes a method for measuring the concentration of EAP using EAP phosphatase and acetaldehyde dehydrogenase.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] As mentioned above, a plasma EAP concentration of 1.5 μM or less is used as a reference value for diagnosing depression. However, the method for measuring EAP concentration using EAP phosphorylase described in Patent Document 2 does not achieve quantitative analysis of trace amounts of EAP. Therefore, a novel measurement method capable of measuring trace amounts of EAP is needed.

[0007] One embodiment of the present invention provides a method for quantifying EAP capable of measuring trace amounts of EAP. Alternatively, one embodiment of the present invention provides a quantitative oxidoreductase for use in the quantitative method. Alternatively, one embodiment of the present invention provides a quantitative composition for use in the quantitative method. Alternatively, one embodiment of the present invention provides a quantitative kit for use in the quantitative method. Alternatively, one embodiment of the present invention provides a sensor chip for use in the quantitative method. Alternatively, one embodiment of the present invention provides a sensor for use in the quantitative method. [Means for solving the problem]

[0008] According to one embodiment of the present invention, a method for quantifying EAP is provided, which includes reacting a sample with a phosphatase to convert EAP contained in the sample to ethanolamine (EA), and then reacting the EA with an oxidoreductase.

[0009] In the method for quantifying EAP, the sample contains at least one amine other than EAP, the oxidoreductase is an oxidoreductase that acts on at least one amine, and the oxidoreductase may be applied to at least one amine before the phosphatase is applied.

[0010] In a method for quantifying EAP, the concentration of EAP may be determined by reducing the mediator by reacting EA with oxidoreductase and then quantifying the reduced mediator.

[0011] In a method for quantifying EAP, the oxidoreductase is an oxidase, and the concentration of EAP may be determined by quantifying the hydrogen peroxide produced by reacting EA with the oxidase.

[0012] In a method for quantifying EAP, the concentration of EAP may be determined by reacting it with oxidase and quantifying the generated hydrogen peroxide or consumed oxygen.

[0013] A method for quantifying EAP may include a first oxidoreductase that acts on EA and a second oxidoreductase that acts on at least one amine.

[0014] In the method for quantifying EAP, at least one of the first oxidoreductase and the second oxidoreductase may be an oxidase.

[0015] According to one embodiment of the present invention, an oxidoreductase is provided which is selected from oxidoreductases belonging to EC number 1.4 or EC number 1.5 and used in a method for quantifying EAP.

[0016] According to one embodiment of the present invention, an oxidoreductase is provided which is selected from primary amine dehydrogenase, monoamine dehydrogenase, diamine dehydrogenase, polyamine dehydrogenase, ethanolamine dehydrogenase, tyramine dehydrogenase, phenylethylamine dehydrogenase, benzylamine dehydrogenase, histamine dehydrogenase, serotonin dehydrogenase, spermine dehydrogenase, spermidine dehydrogenase, β-alanine dehydrogenase, γ-aminobutyric acid (GABA) dehydrogenase, taurine dehydrogenase, cadaverine dehydrogenase, and agmatine dehydrogenase, and is used in a method for quantifying EAP.

[0017] According to one embodiment of the present invention, an oxidase selected from primary amine oxidase, monoamine oxidase, diamine oxidase, polyamine oxidase, ethanolamine oxidase, tyramine oxidase, phenylethylamine oxidase, benzylamine oxidase, histamine oxidase, serotonin oxidase, spermine oxidase, spermidine oxidase, β-alanine oxidase, γ-aminobutyric acid (GABA) oxidase, taurine oxidase, cadaverine oxidase, and agmatine oxidase is provided, which is used in the method for quantifying EAP.

[0018] According to one embodiment of the present invention, a composition for quantifying EAP is provided, which contains phosphatase and an oxidoreductase acting on EA.

[0019] In the composition for quantifying EAP, it may further contain a mediator reduced by adding an oxidoreductase and a reagent reacting with the reduced mediator.

[0020] In the composition for quantifying EAP, the oxidoreductase is an oxidase, and it may further contain a reagent reacting with hydrogen peroxide generated by adding the oxidase.

[0021] According to one embodiment of the present invention, a kit for quantifying EAP is provided, which contains phosphatase and an oxidoreductase acting on EA.

[0022] In the kit for quantifying EAP, it may contain a first reagent containing an oxidoreductase and a second reagent containing a mediator reduced by adding dehydrogenase.

[0023] In the kit for quantifying EAP, as the oxidoreductase, it may contain a first reagent containing an oxidase and a second reagent containing a reagent reacting with hydrogen peroxide generated by adding the oxidase.

[0024] In the kit for EAP quantification, it further includes a third reagent containing an oxidoreductase or an oxidase that acts on at least one amine other than EAP, and the third reagent may be added to the sample before adding the first reagent and the second reagent.

[0025] According to an embodiment of the present invention, there is provided a sensor chip including a working electrode, a counter electrode, and a reference electrode, and a reaction layer disposed on the working electrode, the counter electrode, and the reference electrode, wherein the reaction layer contains an oxidoreductase that acts on EA and is used in the method for quantifying EAP.

[0026] According to an embodiment of the present invention, there is provided a sensor including the above-mentioned sensor chip.

Advantages of the Invention

[0027] According to an embodiment of the present invention, there is provided a method for quantifying EAP capable of measuring trace amounts of EAP. Or, according to an embodiment of the present invention, there is provided an oxidoreductase for quantification used in the quantification method. Or, according to an embodiment of the present invention, there is provided a composition for quantification used in the quantification method. Or, according to an embodiment of the present invention, there is provided a kit for quantification used in the quantification method. Or, according to an embodiment of the present invention, there is provided a sensor chip used in the quantification method. Or, according to an embodiment of the present invention, there is provided a sensor used in the quantification method.

Brief Description of the Drawings

[0028] [Figure 1] It is a flowchart for explaining the method for quantifying EAP according to an embodiment of the present invention. [Figure 2] It is a flowchart for explaining the method for quantifying EAP according to an embodiment of the present invention. [Figure 3] It is a flowchart for explaining the method for quantifying EAP according to an embodiment of the present invention. [Figure 4](a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and (b) to (d) are schematic diagrams showing the components that make up the sensor chip 10. [Figure 5] (a) is a schematic diagram of sensor 100 according to one embodiment of the present invention, and (b) is a block diagram of sensor 100 according to one embodiment of the present invention. [Figure 6] This figure shows the reactivity of LrHP between EAP (ALP(-) in the figure) or EAP reacted with ALP (ALP(+) in the figure) according to one embodiment of the present invention. [Figure 7] (a) is a figure showing the measurement results of an EAP sample containing an interfering amine to which 10 μl of water was added instead of ALP, and (b) is a figure showing the measurement results of a sample to which 10 μl of ALP was added to an EAP sample containing an interfering amine according to one embodiment of the present invention. [Figure 8] This figure shows the results of measuring EAP when the EAP concentration in a sample of one embodiment of the present invention was changed to 1.0 μM, 1.5 μM, 3.0 μM, and 5.0 μM. [Figure 9] This figure shows the results of measuring EAP in one embodiment of the present invention, in which ALP is combined with AgPEAOX, and the results of measuring EAP when the EAP concentration in the sample was changed to 0.2 mM, 0.5 mM, and 1.0 mM. [Modes for carrying out the invention]

[0029] The following describes the quantitative method for EAP, the oxidoreductase for quantification, the quantitative composition, the quantitative kit, the sensor chip, and the sensor according to the present invention. However, the quantitative method for EAP, the oxidoreductase for quantification, the quantitative composition, the quantitative kit, the sensor chip, and the sensor according to the present invention are not limited to the descriptions in the embodiments and examples shown below.

[0030] At the time of filing this application, no known enzyme capable of directly quantifying EAP with high sensitivity was available as a publicly known technique. As a result of our investigations, we found that by combining a phosphatase capable of converting EAP to EA with an oxidoreductase that acts on EA, it is possible to quantify EAP with high sensitivity.

[0031] A method for quantifying EAP according to the present invention includes, in one embodiment, (1) a step of reacting a sample with phosphatase to convert the EAP contained in the sample into EA, and (2) a step of reacting the converted EA with oxidoreductase.

[0032] Figure 1 is a flowchart illustrating a method for quantifying EAP according to one embodiment of the present invention. First, phosphatase is added to the sample and reacted with EAP to convert it to EA (S101). Oxidoreductase is added to the sample and reacted with the converted EA (S103). The reaction product generated by this reaction is detected (S105). Using a calibration curve prepared in advance, EAP is quantified from the detected value of the reaction product (S107).

[0033] In step S101, the EAP concentration in the sample is not particularly limited, but may be, for example, 0.01 μM to 10000 μM. The pH when performing the phosphatase treatment of the sample may be left unadjusted, or it may be adjusted to pH 2 to 13, preferably pH 3 to 12, using a suitable pH buffer to ensure a pH suitable for the action of the phosphatase used. Examples of usable buffers include N-[tris(hydroxymethyl)methyl]glycine, phosphate, acetate, carbonate, tris(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutamate, tricine, HEPES, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, phthalic acid, tartaric acid, etc. The processing temperature may be, for example, 20 to 50°C, or, depending on the phosphatase used, a higher temperature range of 45 to 70°C may be used. The processing time should be sufficient to release EA, and can be 5 seconds to 180 minutes, preferably 0.5 to 60 minutes, more preferably 1 to 30 minutes, and even more preferably 1 to 10 minutes. The resulting processing solution may be used as is, or, if necessary, heated, centrifuged, concentrated, diluted, etc.

[0034] In step S103, the duration of action of the oxidoreductase can be, for example, 5 seconds to 180 minutes, preferably 0.5 minutes to 60 minutes, more preferably 1 minute to 30 minutes, and even more preferably 1 minute to 10 minutes. The action temperature depends on the optimal temperature of the oxidoreductase used, but can be, for example, 20°C to 45°C, and a temperature commonly used in enzymatic reactions can be appropriately selected.

[0035] The preferred amount of oxidoreductase acting on EA used in this invention is, for example, added so that the final concentration is 0.001 U / ml to 500 U / ml, preferably 0.01 U / ml to 100 U / ml. Generally, the lower the concentration of the substrate contained in the sample solution, the higher the final concentration of oxidoreductase added should be. The pH during the reaction should preferably be adjusted using a buffer to a pH suitable for the reaction, taking into account the optimal pH of the oxidoreductase, but it is not limited to any pH at which the reaction can occur. For example, pH 3 to pH 11, preferably pH 5 to pH 9. Examples of usable buffering agents include N-[tris(hydroxymethyl)methyl]glycine, phosphate, acetate, carbonate, tris(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutamate, tricine, HEPES, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricin, Bicine, TAPS, phthalic acid, tartaric acid, etc.

[0036] In step S105, the mediator is reduced by an oxidoreductase acting on EA, and the reduced mediator is reacted with a color-developing or color-degrading reagent. Examples of color-developing or color-degrading substrates used in the present invention include DCIP (2,6-Dichlorophenolindophenol), as well as tetrazolium compounds (Tetrazolium blue, Nitro-tetrazolium blue, Water-soluble tetrazolium (WST)-1, WST-3, WST-4, WST-5, WST-8, WST-9), etc.

[0037] In step S105, hydrogen peroxide is generated by an oxidase acting on EA, and the generated hydrogen peroxide is reacted with a color-developing reagent using a catalytic reaction of peroxidase. As color-developing or color-degrading substrates used in the present invention, examples of color-developing agents include ADPS (N-ethyl-N-sulfopropyl-3-methoxyaniline), ALPS (N-ethyl-N-sulfopropylaniline), TOPS (N-ethyl-N-sulfopropyl-3-methylaniline), ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), DAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline), and ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline). Examples include HDAOS (N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline), MAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine). ADOS, ALOS, and TOOS develop color when condensed with 4-aminoantipyrine. DA-64 and DA-67 do not require 4-aminoantipyrine and develop color when prescribed alone.Furthermore, as colorants, methylene blue, 10-(acetylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(phenylcarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(3-(methylcarboxyamino)-hexamethyl-amino)-phenothiazine, 10-(3-(methylcarboxyamino)-4-methyl-phenyl)-amino)-phenothiazine, 1 Examples of colorants include, but are not limited to, 0-((3-(methylcarboxyaminomethyl)-phenyl)-methylamino)-phenothiazine, 10-(1-naphthaleneamino)-phenothiazine, 10-(methyl)-phenothiazine, 10-(phenylamino)-phenothiazine, 10-(methylamino)-phenothiazine, Azure A, Azure B, Azure C, toluidine blue O, 1,9-dimethyl-3,7-bis(dimethylamino)phenothiazine salt, methylene green or its salts, or their leuco derivatives. The colorant may be added so that the final concentration in the reaction solution is 0.001 mM to 10 mM, for example, 0.005 mM to 2 mM.

[0038] In step S105, the amount of EAP in the sample is determined from the detected value of the reaction product using a pre-prepared calibration curve. In this way, the amount of EAP in the sample can be quantified.

[0039] The sample used in the quantitative analysis method for EAP according to the present invention can be any biological sample that may contain EAP, such as a sample derived from blood, plasma, interstitial fluid, urine, tears, sweat, saliva, etc. The sample may be processed as appropriate. For example, it may be concentrated using a centrifugal concentrator.

[0040] Furthermore, in such quantitative methods, if a sample containing EAP contains other substances that can be acted upon by the oxidoreductase used to quantify EAP, the measured value may be misleading. In particular, the higher the proportion of such interfering substances that the oxidoreductase acts upon, the less accurately the target substance, EAP, can be quantified.

[0041] In one embodiment, the sample may contain at least one amine other than EAP. Generally, clinical samples may contain amines such as EA, β-alanine, and taurine as impurities. In the method for quantifying EAP according to the present invention, an oxidoreductase that acts on EA is used. Therefore, if these amines are present in the sample, the oxidoreductase will also act on these amines, making it difficult to accurately quantify the EA derived from the EAP that should be measured. For this reason, it is preferable to decompose or eliminate amines other than EAP contained in the sample.

[0042] In one embodiment, it is preferable to act on the amines in the sample with at least an amine-acting oxidoreductase before acting on the sample with phosphatase. Figure 2 is a flowchart illustrating a method for quantifying EAP according to one embodiment of the present invention. First, oxidoreductase is added to the sample to decompose or eliminate the amines in the sample (S201). Phosphatase is added to the sample and acts on EAP to convert it to EA (S203). The oxidoreductase added in step S201 acts on the converted EA. The reaction product generated by this reaction is detected (S205). Using a calibration curve prepared in advance, EAP is quantified from the detected value of the reaction product (S207).

[0043] In step S201, by first adding an oxidoreductase that acts on at least amines to the sample, the amines contained in the sample are decomposed or eliminated by the oxidoreductase. The extent to which error-causing substances in the sample affect the measurement value in the quantitative method of EAP according to the present invention depends on the amount of amines such as free EA contained in the sample. When measuring a sample in which the amount of amines such as free EA contained in the sample is small, step S201, which eliminates amines such as free EA beforehand, is not necessary, and the phosphatase can be applied directly to the sample. In that case, it can be inferred that all EA generated in the sample is cleaved from the EAP in the sample by the action of the phosphatase.

[0044] On the other hand, when measuring a sample that contains free amines such as EA, it becomes impossible to distinguish between the EA released from the target substance EAP by the action of phosphatase, resulting in measurement errors. To avoid these measurement errors, it is preferable to eliminate the free amines such as EA from the sample beforehand.

[0045] Furthermore, if an oxidoreductase is selected that has high specificity for EA and substantially no effect on EAP, the target substance for quantification, EAP, will not be eliminated in step S201. However, if the properties of the oxidoreductase used pose a risk of eliminating EAP, the target substance for quantification, in step S201, the erroneous elimination of EAP can be avoided by setting a concentration condition in step S201 that eliminates only amines such as EA and not EAP.

[0046] In step S203, phosphatase is added to the sample and reacted with EAP to convert it to EA. Step S203 is the same as step S101 described above, so a detailed explanation is omitted.

[0047] In step S205, the oxidoreductase added in step S201 acts on the EA converted in step S203. The reaction product generated by this reaction can be detected. Step S205 is the same as step S103 except that the already added oxidoreductase acts on the EA, and it is the same as step S105 in that it detects the reaction product generated in the redox reaction, so a detailed explanation is omitted.

[0048] In step S207, EAP is quantified from the detected value of the reaction product using a pre-prepared calibration curve. Since step S207 is the same as step S107, a detailed explanation is omitted.

[0049] In the embodiments described above, an example was explained in which the action of oxidoreductase added in the amine elimination step was directly utilized. However, the method for quantifying EAP according to the present invention is not limited to this. Oxidoreductase may be added during the amine elimination step, and further oxidoreductase may be added during the EAP quantification step.

[0050] Figure 3 is a flowchart illustrating a method for quantifying EAP according to one embodiment of the present invention. First, a first oxidoreductase is added to the sample to decompose or eliminate the amine contained in the sample (S301). A phosphatase is added to the sample and reacted with EAP to convert it to EA (S303). A second oxidoreductase is added to the sample and reacted with the converted EA (S305). The reaction product generated by this reaction is detected (S307). Using a calibration curve prepared in advance, EAP is quantified from the detected value of the reaction product (S309).

[0051] In step S301, the oxidoreductase used to eliminate free amines such as EA in the sample, and in step S305, the oxidoreductase used to quantify the EA released from EAP, the target substance for quantification, by the action of phosphatase, may be of different types or the same type. Furthermore, at least one of the oxidoreductases used to eliminate free amines such as EA in the sample and the oxidoreductase used to quantify the EA released from EAP, the target substance for quantification, by the action of phosphatase, may be an oxidase as described later. The concentrations of enzymes suitable for elimination, suitable for quantification, and substrate specificity of the enzymes used can be appropriately set.

[0052] Step S303 may be the same as step S101 described above. Also, step S305 may be the same as step S103 described above. Step S307 may be the same as step S105 described above. Step S309 may be the same as step S107 described above, and a detailed explanation is omitted.

[0053] <Phosphatase> Any phosphatase can be used in the quantitative analysis method for EAP according to the present invention, as long as it is suitable for use in clinical testing and can effectively cleave EA from EAP in the sample being processed. Examples of such phosphatases include acid phosphatases, alkaline phosphatases, and protein phosphatases. These phosphatases may be used individually or in combination of two or more types.

[0054] In the present invention, when selecting a phosphatase, it is possible to select an enzyme that efficiently cleaves EA from EAP in the sample while simultaneously suppressing the generation of contaminating substances as much as possible. That is, by predicting various phosphate-containing compounds other than the target substance in the sample as contaminating substances and selecting a phosphatase that is less likely to generate them, the generation of contaminating substances can be suppressed. Phosphatases can also be selected based on knowledge regarding which structural bonds various known phosphatases are prone to cleaving.

[0055] In practice, the quantitative analysis method for EAP according to the present invention can also be controlled by selecting the substrate specificity of the oxidoreductase in the step of action by oxidoreductase following cleavage by phosphatase, and by making such a selection, it is expected that the influence of false reactions will be reduced.

[0056] The phosphatase treatment conditions for the sample can be any conditions that allow the phosphatase used to act on EAP and efficiently release EA in a short time. The amount of phosphatase used is appropriately selected depending on the EAP content in the sample and the treatment conditions. For example, phosphatase can be added so that the final concentration is 0.001 U / ml to 10000 U / ml, preferably 0.01 U / ml to 1000 U / ml. Other enzymes may also be added as needed.

[0057] The activity of the phosphatase usable in this invention is calculated by measuring the amount of 4-nitrophenol (maximum absorption wavelength 405 nm) produced by the hydrolysis of 4-nitrophenyl phosphate using a spectrophotometer. As an example, when using alkaline phosphatase (ALP), 1 U was defined as the amount of enzyme that hydrolyzes 1 μmol of 4-nitrophenyl phosphate per minute at 37°C and pH 9.8 (diethanolamine buffer).

[0058] <Oxidoreductase> The oxidoreductase usable in this invention is an enzyme that acts on EA or substrates structurally similar to EA. In this invention, the oxidoreductase used can be replaced with a dehydrogenase that efficiently acts on substrates having a structure similar to EA. A similar structure refers to a physicochemical structure that is considered similar from structural, electronic, stereochemical, or other viewpoints.

[0059] For example, substrates structurally similar to EA include substrates having CH-NH2 or CH-NH bonds, and enzymes of such substrates include oxidoreductases belonging to EC number 1.4 or EC number 1.5.

[0060] For example, oxidoreductases belonging to EC number 1.4 or EC number 1.5 include primary amine dehydrogenases, monoamine dehydrogenases, diamine dehydrogenases, polyamine dehydrogenases, ethanolamine dehydrogenases, tyramine dehydrogenases, phenylethylamine dehydrogenases, benzylamine dehydrogenases, histamine dehydrogenases, serotonin dehydrogenases, spermine dehydrogenases, spermidine dehydrogenases, β-alanine dehydrogenases, γ-aminobutyric acid (GABA) dehydrogenases, taurine dehydrogenases, cadaverine dehydrogenases, and agmatine dehydrogenases. In particular, taurine is a substrate structurally similar to EAP, and taurine dehydrogenase (TDH) can be suitably used as the dehydrogenase for this substrate.

[0061] The oxidoreductase used in the present invention may be a polymer or a monomer. For example, if a certain subunit (monomer) among several subunits constituting a polymeric oxidoreductase catalyzes a dehydrogenation reaction in which hydrogen is taken from a substrate and passed to a hydrogen acceptor, the oxidoreductase used in the present invention may be a polymer or that subunit (monomer).

[0062] In one embodiment, the oxidoreductase used in the present invention has a large subunit (LaTDH) having the base sequence of SEQ ID NO: 1 and a small subunit (SmTDH) having the base sequence of SEQ ID NO: 2. Not only does the polymer containing LaTDH and SmTDH catalyze the dehydrogenation reaction in which hydrogen is removed from a substrate and passed to a hydrogen acceptor, but LaTDH alone also catalyzes the dehydrogenation reaction. Therefore, the oxidoreductase used in the present invention may contain LaTDH and SmTDH, or it may consist of LaTDH alone.

[0063] Furthermore, oxidoreductases belonging to EC number 1.4 or EC number 1.5 may also be oxidases belonging to EC number 1.4.3 or EC number 1.5.3.

[0064] For example, oxidases belonging to EC number 1.4.3 or EC number 1.5.3 include primary amine oxidases, monoamine oxidases, diamine oxidases, polyamine oxidases, ethanolamine oxidases, tyramine oxidases, phenylethylamine oxidases, benzylamine oxidases, histamine oxidases, serotonin oxidases, spermine oxidases, spermidine oxidases, β-alanine oxidases, γ-aminobutyric acid (GABA) oxidases, taurine oxidases, cadaverine oxidases, and agmatine oxidases. In particular, phenylethylamine oxidase (PEAOX) can be preferably used.

[0065] The reaction conditions for the oxidoreductase used in the quantitative analysis method for EAP according to the present invention can be any conditions that act on EA and efficiently catalyze the oxidation reaction. However, enzymes generally have an optimal temperature and pH at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperature and pH are preferable. For example, the reaction conditions for oxidoreductase can preferably be 30°C and pH 8.5, as described later, but are not limited to these. Similarly, for example, the reaction conditions for PEAOX can preferably be 37°C and pH 8.5, as described later, but are not limited to these.

[0066] The oxidoreductase used in the EAP quantification method according to the present invention may be an oxidoreductase produced by naturally occurring microorganisms or an oxidoreductase produced by a transformed microorganism. From the viewpoint of efficient high-volume expression of the enzyme, using a transformed microorganism allows for efficient high-volume expression of the enzyme.

[0067] The present quantitative method of EAP is based on microorganisms derived from microorganisms, special limitations, and paracoccus. genus, Methylarcula genus, Martelella genus, Rhodobacter genus, Roseobacter genus, Gemmo bacter, Arthrobacter, Paenarthrobacter, Pseudarthrobacter, Cryobacterium, Bacillus, Sinomonas, Tersicoccus, Kocuria, M *Icrococcus*, *Brevibacterium*, *Zhihengliuella*, *Citricoccus*, *Geodematophilus*, *Rhodococcus*, *Amycolatopsis*, *Nocardia*, *Modestobacter*, *Glutamicibacter*, *Psudonocardia*, *Gordonia*, *Streptomyces*, *Geodermatophilus*, *Cellulomonas*, *Mycobacterium*, *Mycolicibacterium*, *Psudoglutamicibacter*, *Corynebacterium*, *Nocardiopsis*, *Nonomuraea*, *Saccharomonospora*, *Prauserella*, *Amnibacterium*, *Actinobacteria*, *Saccharopolyspora*, *Leifsonia*, *Agromyces*, *Streptacidiphilus*, *Xylanimonas*, *Tsukamurella*, *Williams* ia, Asanoa, Plantactinospora, Salinispora, Agreia, Cryocola, Curtobacterium, Murinocardiopsis, Subtercola, Microbispora, Jiangella, Blastococcus, Actinomadura, Actinoplanes, Catenulispora, Lichtheimia, Syncephalastrum

[0068] For example, the oxidoreductase used in the EAP quantification method according to the present invention may be TDH produced by Paracoccus denitrigicans, or TDH produced by Escherichia coli transformed with a plasmid containing the TDH gene derived from Paracoccus denitrigicans. However, by using Escherichia coli transformed with a plasmid containing the TDH gene derived from Paracoccus denitrigicans, oxidoreductase can be expressed in large quantities efficiently.

[0069] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be PEAOX produced by Arthrobacter globiformis, or PEAOX produced by Escherichia coli transformed with a plasmid containing the PEAOX gene derived from Arthrobacter globiformis. However, by using Escherichia coli transformed with a plasmid containing the PEAOX gene derived from Arthrobacter globiformis, oxidoreductase can be expressed in large quantities efficiently.

[0070] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be amine oxidase (LcAOX) produced by Lichtheimia corymbifera, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the LcAOX gene having the base sequence of Sequence ID No. 3 derived from Lichtheimia corymbifera. However, by using Escherichia coli transformed with a plasmid containing the LcAOX gene derived from Lichtheimia corymbifera, oxidoreductase can be expressed in large quantities efficiently.

[0071] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be the hypothetical protein (LrHP) produced by Lichtheimia ramosa, or the hypothetical protein produced by Escherichia coli transformed with a plasmid containing the LrHP gene having the base sequence of Sequence ID No. 4 derived from Lichtheimia ramosa. However, by using Escherichia coli transformed with a plasmid containing the LrHP gene derived from Lichtheimia ramosa, oxidoreductase can be expressed in large quantities efficiently.

[0072] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be amine oxidase (SrAOX3925) produced by Syncephalastrum racemosum, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the SrAOX3925 gene having the base sequence of Sequence ID No. 5 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrAOX3925 gene derived from Syncephalastrum racemosum, oxidoreductase can be efficiently expressed in large quantities.

[0073] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be amine oxidase (SrAOX3926) produced by Syncephalastrum racemosum, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the SrAOX3926 gene having the base sequence of Sequence ID No. 6 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrAOX3926 gene derived from Syncephalastrum racemosum, oxidoreductase can be efficiently expressed in large quantities.

[0074] Furthermore, for example, the oxidoreductase used in the EAP quantification method according to the present invention may be ethanolamine oxidase (SrEAOX) produced by Syncephalastrum racemosum, or ethanolamine oxidase produced by Escherichia coli transformed with a plasmid containing the SrEAOX gene having the nucleotide sequence of Sequence ID No. 7 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrEAOX gene derived from Syncephalastrum racemosum, oxidoreductase can be efficiently expressed in large quantities.

[0075] In one embodiment, the oxidoreductase used in the quantitative method of EAP according to the present invention is Paracoccus Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of LaTDH produced by denitrigicans (SEQ ID NO: 8), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0076] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Arthrobacter Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of PEAOX produced by globiformis (SEQ ID NO: 9), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0077] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Lichtheimia Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of LcAOX produced by corymbifera (SEQ ID NO: 10), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0078] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Lichtheimia Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of LrHP produced by ramosa (SEQ ID NO: 11), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0079] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Syncephalastrum Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of SrAOX3925 produced by racemosum (SEQ ID NO: 12), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0080] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Syncephalastrum Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of SrAOX3926 produced by racemosum (SEQ ID NO: 13), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0081] In one embodiment, the oxidoreductase used in the quantitative analysis method of EAP according to the present invention is Syncephalastrum Examples include oxidoreductases having high sequence identity (e.g., 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more) with respect to the amino acid sequence of SrEAOX produced by racemosum (SEQ ID NO: 14), and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0082] (Identity of amino acid sequence) The identity of amino acid sequences can be calculated using programs such as GENETYX® (Genetics Corporation)'s Maximum Matching and Search Homology, or DNASIS® Pro (Hitachi Solutions, Ltd.)'s Maximum Matching and Multiple Alignment, or CLUSTAL W's Multiple Alignment. To calculate the identity of amino acid sequences, when the amino acid sequences of two or more oxidoreductases are aligned, the positions of identical amino acids in those two or more oxidoreductases can be examined. Based on this information, identical regions in the amino acid sequences can be determined. Here, for two or more amino acid sequences, identity % refers to the percentage obtained when the total number of amino acids in the alignable region is used as the denominator, and the number of positions occupied by identical amino acids is used as the numerator, when the alignment of two or more amino acid sequences is performed using an algorithm such as Blosum62. Therefore, if there are regions in two or more amino acid sequences that show no identity whatsoever, for example, if one of the amino acid sequences has an additional sequence at the N-terminus or C-terminus that shows no identity whatsoever, then those regions that lack identity are impossible to align and are therefore not used in calculating the identity percentage.

[0083] (Method for preparing enzymes) The following describes a method for preparing oxidoreductase used in the quantitative analysis of EAP according to the present invention.

[0084] (Construction of expression plasmids) The plasmid for oxidoreductase expression used in the EAP quantification method according to the present invention is obtained by a commonly used method. For example, DNA is extracted from a microorganism that produces oxidoreductase, and a DNA library is prepared. From the prepared DNA library, the DNA fragment encoding the oxidoreductase according to the present invention is identified and isolated. Using complementary primers with the isolated DNA fragment as a template, the DNA fragment is amplified by polymerase chain reaction (PCR), and the gene encoding the oxidoreductase is cloned. The amplified DNA fragment is ligated to a vector to obtain a plasmid having the DNA fragment encoding the oxidoreductase according to the present invention.

[0085] Alternatively, a DNA fragment encoding oxidoreductase can be chemically synthesized, and this DNA fragment can be ligated to a vector to obtain a plasmid containing DNA encoding oxidoreductase.

[0086] The obtained plasmid is used to transform strains of E. coli and other bacteria to obtain strains of E. coli and other bacteria that have DNA encoding oxidoreductase.

[0087] Furthermore, for example, a yeast strain may be transformed using the obtained plasmid to obtain a yeast strain having DNA encoding oxidoreductase. Suitable methods for transforming yeast include known methods such as lithium acetate (MethodsMol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484), but are not limited to these. Transformation can be performed using any method, including spheroplast methods and glass bead methods. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The plasmid containing oxidoreductase-encoding DNA may also contain marker genes to enable the selection of transformed cells. Examples of marker genes include genes that complement the host's nutritional requirements, such as URA3 and TRP1. Furthermore, it is desirable that the plasmid containing the DNA encoding the oxidoreductase includes a promoter or other regulatory sequence (e.g., a secretion signal sequence, an enhancer sequence, a terminator sequence, or a polyadenylation sequence) that enables the expression of the oxidoreductase gene of the present invention in host cells. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter.

[0088] Furthermore, other examples of host cells include filamentous fungi such as those of the genera Aspergillus and Tricoderma. The method for producing transformants of filamentous fungi is not particularly limited, and for example, one method is to insert the DNA encoding oxidoreductase into the host filamentous fungus in a manner that expresses it, according to a conventional method. Specifically, a DNA construct is prepared by inserting the gene encoding the oxidoreductase of the present invention between an expression-inducing promoter and a terminator, and then the host filamentous fungus is transformed with the DNA construct containing the gene encoding oxidoreductase to obtain a transformant that overexpresses the gene encoding oxidoreductase. In this specification, a DNA fragment consisting of an expression-inducing promoter, a gene encoding oxidoreductase, and a terminator, and a recombinant vector containing said DNA fragment, prepared for transforming a host filamentous fungus, are collectively referred to as a DNA construct.

[0089] The method for inserting the gene encoding oxidoreductase into a host filamentous fungus in a manner in which it is expressed is not particularly limited, but examples include methods of directly inserting it onto the chromosome of the host organism using homologous recombination, or methods of introducing it into the host filamentous fungus by linking it onto a plasmid vector.

[0090] In methods utilizing homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of the host fungus. By overexpressing the construct within the host fungus under the control of its own high-expression promoter, a transformant can be obtained through self-cloning. The high-expression promoter is not particularly limited, but examples include the promoter region of the translation elongation factor TEF1 gene (tef1), the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0091] In the vector-based method, a DNA construct can be incorporated into a plasmid vector used for filamentous fungal transformation using a standard method, and the corresponding host filamentous fungus can then be transformed using a standard method.

[0092] Such suitable vector-host systems are not particularly limited as long as they are capable of producing the oxidoreductase of the present invention in the host filamentous fungus, and include, for example, the pUC19 and filamentous fungus system, and the pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and filamentous fungus system.

[0093] While it is preferable to introduce the DNA construct into the chromosome of the host filamentous fungus, another method is to incorporate the DNA construct into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)), thereby allowing it to be used without being introduced into the chromosome.

[0094] The DNA construct may include marker genes to enable the selection of transformed cells. The marker genes are not particularly limited and include, for example, genes that complement the host's nutritional requirements, such as pyrG, niaD, and adeA; and drug resistance genes to drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably includes promoters, terminators, and other regulatory sequences (e.g., enhancers, polyadenylation sequences) that enable the overexpression of the gene encoding the oxidoreductase of the present invention in host cells. The promoters are not particularly limited but include suitable inductive and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminators are also not particularly limited but include, for example, the alp terminator, amy terminator, and tef1 terminator.

[0095] In a DNA construct, the gene expression regulatory sequence of the gene encoding oxidoreductase is not necessarily required if the DNA fragment containing the gene encoding the oxidoreductase of the present invention to be inserted contains a sequence that has an expression regulatory function. Furthermore, when transformation is performed by cotransformation, the DNA construct may not need to contain a marker gene.

[0096] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, the gene encoding oxidoreductase, the alp gene terminator, and the pyrG marker gene are ligated to the In-Fusion Cloning Site in the multi-cloning site of pUC19.

[0097] As a method for transforming filamentous fungi, a method known to those skilled in the art can be appropriately selected. For example, after preparing protoplasts of the host filamentous fungus, the protoplast PEG method using polyethylene glycol and calcium chloride can be used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, Japanese Patent Publication No. 2007-222055). The culture medium for regenerating the transformed filamentous fungus should be appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, if Aspergillus soybean is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the regeneration of the transformed filamentous fungus can be carried out, for example, in Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2M sorbitol.

[0098] (Enzyme recombinant expression) A bacterial strain, such as Escherichia coli, having DNA encoding oxidoreductase for use in the quantitative analysis method of EAP according to the present invention, is cultured in a culture medium. When culturing the microbial host cells, the culture can be carried out by aerated stirring deep culture, shaking culture, static culture, etc., at a culture temperature of 10°C to 42°C, preferably at around 25°C for several hours to several days, and more preferably at around 25°C for 1 to 7 days. As the culture medium for culturing the above microbial host cells, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or extract of soybeans or wheat bran are added to one or more inorganic salts such as sodium chloride, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate, and carbohydrate raw materials, vitamins, etc. are added as needed. The bacterial cells are separated from the culture solution obtained by culturing by centrifugation. The isolated bacterial cells are subjected to ultrasonic pulverization, grinding, or other methods, or treated with lytic enzymes such as lysozyme or yatarase to form a suspension. A crude enzyme solution is then obtained from the fraction obtained by centrifugation of the suspension.

[0099] (Purification of oxidoreductase) Any method that can purify the enzyme from the crude enzyme solution is acceptable for purifying oxidoreductase. For example, oxidoreductase can be purified from the crude enzyme solution by commonly used methods such as ion exchange chromatography and gel filtration chromatography.

[0100] (Measurement of oxidoreductase activity) Any method for measuring oxidoreductase activity is acceptable, as long as it directly or indirectly measures the products of the redox reaction catalyzed by oxidoreductase. For example, enzyme activity can be measured by measuring the current generated when a reduced product is produced by oxidoreductase catalyzing a redox reaction and the reduced product transfers electrons to an electrode. Preferably, enzyme activity can be measured by reacting the reduced product from the redox reaction catalyzed by oxidoreductase with a reagent containing an absorbent substance that reacts with the reduced product (hereinafter referred to as "absorbent reagent") and measuring the absorbance.

[0101] (Quantitative composition for EAP and quantitative kit for EAP) The method for quantifying EAP using phosphatase and oxidoreductase according to the present invention may be carried out by providing a composition containing phosphatase and oxidoreductase acting on EA, or by combining phosphatase, oxidoreductase acting on EA, and a commercially available product reaction reagent. For example, the method may be provided as a quantification composition for EAP containing phosphatase and oxidoreductase acting on EA, or as a quantification composition for EAP further containing a mediator that is reduced by the addition of oxidoreductase and a reagent that reacts with the reduced mediator.

[0102] In one embodiment, a kit for quantifying EAP may be provided, comprising a phosphatase, an oxidoreductase, a mediator that is reduced by the addition of the oxidoreductase, and a reagent that reacts with the reduced mediator. For example, the kit for quantifying EAP may comprise a first reagent comprising a phosphatase and an oxidoreductase, and a second reagent comprising a mediator that is reduced by the addition of the oxidoreductase. It may further comprise a third reagent comprising an oxidoreductase or oxidase that acts on at least one amine other than ethanolamine phosphate, and the third reagent may be added to the sample before adding the first and second reagents.

[0103] The mediator (also called an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the quantitative method or quantitative kit of the present invention is not particularly limited as long as it can accept electrons from oxidoreductase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides, such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and their derivatives, and examples of phenazine compounds include, but are not limited to, 5-methylphenazinium methosulfate (PMS) and methoxy-PMS.

[0104] (Sensor chip and electrodes) Figure 4(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and Figures 4(b) to 4(d) are schematic diagrams showing the components constituting the sensor chip 10. The sensor chip 10 comprises two or more electrodes arranged on a substrate 11. The substrate 11 is made of an insulating material. In Figures 4(a) and 4(b), as an example, an working electrode 1, a counter electrode 3, and a reference electrode 5 are arranged on the substrate 11. Each electrode is electrically connected to a wiring section 7, and the wiring section 7 is electrically connected to a terminal 9 located on the opposite side of the wiring direction from each electrode. The working electrode 1, counter electrode 3, and reference electrode 5 are spaced apart from each other. Furthermore, it is preferable that the working electrode 1, counter electrode 3, and reference electrode 5 are integrally formed with the wiring section 7 and the terminal 9. Alternatively, the counter electrode 3 and the reference electrode 5 may be an integrated type.

[0105] As shown in Figures 4(a) and 4(c), a spacer 13 is placed at the end of the base material 11 parallel to the wiring section 7, and a cover 15 covering the working electrode 1, counter electrode 3, reference electrode 5, and spacer 13 is placed thereon. The spacer 13 and cover 15 are made of insulating material. The spacer 13 preferably has a thickness approximately equal to that of the working electrode 1, counter electrode 3, and reference electrode 5, and is in close contact with the working electrode 1, counter electrode 3, and reference electrode 5. Alternatively, the spacer 13 and cover 15 may be formed as a single unit. The cover 15 is a protective layer that prevents the wiring section 7 from deteriorating due to exposure to the outside air and prevents short circuits due to seepage of the measurement sample.

[0106] In one embodiment, the oxidoreductase of the present invention may be coated, adsorbed, or immobilized on an electrode. Preferably, the oxidoreductase of the present invention is coated, adsorbed, or immobilized on the working electrode. In another embodiment, a phosphatase may also be coated, adsorbed, or immobilized on the electrode together with the oxidoreductase. In yet another embodiment, a mediator may also be coated, adsorbed, or immobilized on the electrode together with the oxidoreductase and phosphatase. The oxidoreductase, or the oxidoreductase and phosphatase, or the oxidoreductase, phosphatase and mediator may be contained in the reaction layer 19 arranged on the working electrode 1, the counter electrode 3, and the reference electrode 5. As electrodes, carbon electrodes, metal electrodes such as platinum, gold, silver, nickel, and palladium can be used. In the case of carbon electrodes, examples of materials include pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, and plastic-formed carbon (PFC). The measurement system may be a two-electrode system or a three-electrode system, and for example, the enzyme can be immobilized on the working electrode. Examples of reference electrodes include standard hydrogen electrodes, reversible hydrogen electrodes, silver-silver chloride electrodes (Ag / AgCl), palladium-hydrogen electrodes, and saturated calomel electrodes, but from the viewpoint of stability and reproducibility, it is preferable to use Ag / AgCl.

[0107] Oxidoreductase can be immobilized on electrodes by methods such as crosslinking, coating with a dialysis membrane, encapsulation in a polymer matrix, use of photocrosslinkable polymers, use of electrically conductive polymers, or use of oxidation / reduction polymers. Alternatively, the enzyme may be immobilized together with a mediator in a polymer or adsorbed onto the electrode, and these methods may be combined.

[0108] The mediator (also called an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the composition, kit, electrode, or sensor chip of the present invention is not particularly limited as long as it can accept electrons from oxidoreductase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides, such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and their derivatives, and examples of phenazine compounds include, but are not limited to, PMS and methoxyPMS.

[0109] The oxidoreductase of the present invention can be applied to various electrochemical measurement techniques by using a potentiostat or galvanostat. Examples of electrochemical measurement techniques include amperometry, potentiometry, and coulometry. For example, using amperometry, the concentration of EAP in a sample can be calculated by measuring the current generated when hydrogen peroxide, produced when oxidoreductase reacts with EAP, is applied to a hydrogen peroxide electrode at a voltage of +600mV to +1000mV (vs. Ag / AgCl). For example, a calibration curve can be created by measuring the current values ​​for known EAP concentrations (0μM, 0.1μM, 0.15μM, 0.3μM, 0.5μM, 50μM, 100μM, 150μM, 200μM) and plotting them against the EAP concentration. The EAP concentration can then be obtained from the calibration curve by measuring the current value for an unknown EAP. For example, a carbon electrode or a platinum electrode can be used as the hydrogen peroxide electrode. Alternatively, instead of a hydrogen peroxide electrode, an electrode immobilized with reductases such as peroxidase or catalase can be used. By applying -400mV to +100mV (vs. Ag / AgCl) and measuring the resulting reduction current, the amount of hydrogen peroxide can be quantified, and the EAP value can be measured.

[0110] Furthermore, by mixing a mediator into the reaction solution and, for example, using amperometry, transferring electrons generated when oxidase reacts with EAP to an oxidized mediator to produce a reduced mediator, and then applying -1000mV to +500mV (vs. Ag / AgCl), the current value generated can be measured to calculate the concentration of EAP in the sample. A carbon electrode or platinum electrode is preferred as the counter electrode. For example, a calibration curve can be created by measuring the current value for known EAP concentrations (0μM, 0.1μM, 0.15μM, 0.3μM, 0.5μM, 50μM, 100μM, 150μM, 200μM) and plotting it against the EAP concentration. The EAP concentration can be obtained from the calibration curve by measuring the current value for an unknown EAP.

[0111] Furthermore, printed electrodes (sensor chips) can be used to reduce the amount of solution required for measurement. In this case, it is preferable that the electrodes are formed on a substrate made of an insulating substrate. Specifically, it is desirable that the electrodes be formed on the substrate by printing techniques such as photolithography, screen printing, gravure printing, and flexographic printing. Examples of insulating substrate materials include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material that has strong resistance to various solvents and chemicals.

[0112] [EAP Measurement Sensor] In one embodiment, an EAP measurement sensor using the oxidoreductase of the present invention is provided. Figure 5(a) is a schematic diagram of a sensor 100 according to one embodiment of the present invention. The sensor is an EAP measurement device using the oxidoreductase of the present invention, and comprises a sensor chip containing the oxidoreductase and a measurement unit. The measurement unit 30 may include, for example, a switch 31 which is an input unit and a display 33 which is a display unit. The switch 31 may be used, for example, to control the ON / OFF of the power supply of the measurement unit 30, or to control the start and interruption of EAP measurement at the sensor 100. The display 33 may, for example, display the measured value of EAP, and may include a touch panel as an input unit for controlling the measurement unit 30.

[0113] Figure 5(b) is a block diagram of a sensor 100 according to one embodiment of the present invention. The sensor 100 may include, for example, a control unit 110, a display unit 120, an input unit 130, a storage unit 140, a communication unit 150, and a power supply 160 in the measurement unit 30, and these may be electrically connected to each other by wiring 190. In addition, the terminals of the sensor chip 10, which will be described later, and the terminals of the measurement unit 30 are electrically connected, and the current generated in the sensor chip 10 is detected by the control unit 110. The control unit 110 is a control device that controls the sensor 100, and consists of, for example, a known central processing unit (CPU) and an operation program that controls the sensor 100. Alternatively, the control unit 110 may include a central processing unit and an operating system (OS), and may include an application program or module for performing EAP measurement.

[0114] The display unit 120 may include, for example, a known display 33, and may display the EAP measurement value, the status of the measurement unit 30, or operation requests to the operator. The input unit 130 is an input device for the operator to operate the sensor 100, and may be, for example, a switch 31 or a touch panel located on the display 33. Multiple switches 31 may be located on the measurement unit 30.

[0115] The storage unit 140 consists of a main memory, and an auxiliary storage device (hard disk) may be located externally. The main memory may consist of read-only memory (ROM) and / or random access memory (RAM). Operation programs, operating systems, application programs, or modules are stored in the storage unit 140 and executed by the central processing unit to constitute the control unit 110. Measured values ​​and current values ​​can also be stored in the storage unit 140.

[0116] The communication unit 150 is a known communication device that connects the sensor 100 or the measuring unit 30 to an external device (computer, printer, or network). The communication unit 150 and the external device are connected by wired or wireless communication. The power supply 160 is a known power supply device that supplies power to the sensor 100 or the measuring unit 30.

[0117] As described above, the present invention provides a method for quantifying EAP, an oxidoreductase for quantification, a composition for quantification, and a kit for quantification, which contain phosphatase and oxidoreductase, thereby providing a novel method for quantifying EAP concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, and a sensor for quantification, which are biomarkers for depression.

[0118] (Method for quantifying EAP using oxidase activity) The method for quantifying EAP using oxidase activity according to this embodiment involves reacting a sample with phosphatase to convert the EAP contained in the sample into EA, reacting the hydrogen peroxide produced by reacting EA with oxidase with a reagent, and determining the concentration of EAP.

[0119] Refer to Figure 1. First, phosphatase is added to the sample and reacted with EAP to convert it to EA (S101). Oxidase is added to the sample and reacted with the converted EA (S103). The hydrogen peroxide produced by this reaction is detected (S105). Using a previously prepared calibration curve, EAP is quantified from the detected hydrogen peroxide value (S107).

[0120] Since step S101 has been described above, a detailed explanation will be omitted. In step S103, the EAP concentration in the sample is not particularly limited, but may be, for example, 0.01 μM to 10000 μM. The reaction time can be, for example, 5 seconds to 180 minutes, preferably 0.5 minutes to 60 minutes, more preferably 1 minute to 30 minutes, and even more preferably 1 minute to 10 minutes. The reaction temperature depends on the optimal temperature of the enzyme used, but may be, for example, 20°C to 45°C, and a temperature commonly used in enzyme reactions can be appropriately selected.

[0121] A suitable amount of oxidase to use is, for example, to add it so that the final concentration is 0.001 U / ml to 500 U / ml, preferably 0.01 U / ml to 100 U / ml. Generally, the lower the concentration of the substrate in the sample solution, the higher the final concentration of oxidase to be added. The pH when reacting with oxidase should preferably be adjusted using a buffer to a pH suitable for the reaction, taking into account the optimal pH of oxidase, but it is not limited to this as long as the pH is suitable for the reaction. For example, the pH when reacting with oxidase is pH 3 to pH 11, preferably pH 5 to pH 9. The buffers that can be used are the same as those used when quantifying EAP with oxidoreductase.

[0122] The present invention provides a method for measuring EAP by measuring the products or consumptions resulting from the action of oxidase acting on EA. Hydrogen peroxide is a preferred product to measure because it is easy to measure. Hydrogen peroxide generated by the action of oxidase may also be detected using a chromogenic substrate. Examples of chromogenic substrates used in the present invention include, in addition to 4-aminoantipyrine, ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine). ADOS, ALOS, and TOOS develop color when condensed with 4-aminoantipyrine. DA-64 and DA-67 do not require 4-aminoantipyrine and develop color when prescribed alone. In all cases, the color reaction is catalyzed by peroxidase. Dissolved oxygen is one of the consumables to be measured, and the amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like. For example, the degree of color development (change in absorbance) of the above-mentioned reagents can be measured using a spectrophotometer or an automated biochemical analyzer, and the amount of EAP contained in the sample can be measured by comparing it with the absorbance of a standard sample.

[0123] In step S105, the amount of EAP in the sample is determined from the detected value of the reaction product using a pre-prepared calibration curve. In this way, the amount of EAP in the sample can be quantified.

[0124] Examples of samples used in the quantitative analysis method for EAP according to the present invention include any biological sample that may contain EAP, such as samples derived from blood, plasma, etc. The sample may be processed as appropriate. The sample may be concentrated, for example, by a centrifugal concentrator.

[0125] However, in such quantitative methods, if a sample containing EAP contains other substances that can be acted upon by the oxidase used to quantify EAP, the measured value may be misleading. In particular, the higher the proportion of such interfering substances that the oxidase acts upon, the less accurately the target substance, EAP, can be quantified.

[0126] In one embodiment, the sample may contain at least one amine other than EAP. Generally, clinical samples may contain amines such as EA, β-alanine, and taurine as impurities. In the method for quantifying EAP according to the present invention, an oxidase that acts on EA is used, so if these amines are present in the sample, the oxidase will also act on these amines, making it difficult to accurately quantify the EA derived from the EAP that should be measured. For this reason, it is preferable to decompose or eliminate amines other than EAP contained in the sample.

[0127] In one embodiment, it is preferable to act on the amines in the sample with at least an amine-acting oxidase before acting on the sample with phosphatase. See Figure 2. First, oxidase is added to the sample to decompose or eliminate the amines in the sample (S201). Phosphatase is added to the sample and acts on EAP to convert it to EA (S203). The oxidase added in step S201 acts on the converted EA. The hydrogen peroxide produced by this reaction is detected (S205). Using a pre-prepared calibration curve, EAP is quantified from the detected hydrogen peroxide value (S207).

[0128] In step S201, by first adding an oxidase that acts on at least amines to the sample, the amines contained in the sample are decomposed or eliminated by the oxidase. The extent to which error-causing substances in the sample affect the measurement value in the quantitative analysis method of EAP according to the present invention depends on the amount of free EA contained in the sample. When measuring a sample containing a small amount of free EA, step S201, which eliminates the free EA beforehand, is not necessary, and the phosphatase can be applied directly to the sample. In that case, it can be inferred that all the EA generated in the sample is cleaved from the EAP in the sample by the action of the phosphatase.

[0129] On the other hand, when measuring a sample that contains free EA, it becomes impossible to distinguish between the EA released from the target substance EAP by the action of phosphatase and the free EA, resulting in measurement errors. To avoid these measurement errors, it is preferable to eliminate the free EA in the sample beforehand.

[0130] Furthermore, if an oxidase is selected that has high specificity for EA and virtually no effect on EAP, then in step S201, the target substance for quantification, EAP, will not be eliminated. However, if the properties of the oxidase used pose a risk of eliminating EAP, the target substance for quantification, in step S201, then the erroneous elimination of EAP can be avoided by setting a concentration condition that eliminates only EA and not EAP, and then performing step S201.

[0131] In step S203, phosphatase is added to the sample and reacted with EAP to convert it to EA. Step S203 is the same as step S101 described above, so a detailed explanation is omitted.

[0132] In step S205, the oxidase added in step S201 acts on the EA converted in step S203. The reaction product generated by this reaction can be detected. Step S205 is the same as step S103 except that the already added oxidase acts on the EA, and it is the same as step S105 in that it detects the reaction product generated in the redox reaction, so a detailed explanation is omitted.

[0133] In step S207, EAP is quantified from the detected value of the reaction product using a pre-prepared calibration curve. Since step S207 is the same as step S107, a detailed explanation is omitted.

[0134] In the embodiments described above, an example was explained in which the action of oxidase added in the amine elimination step was directly utilized. However, the quantitative determination method of EAP according to the present invention is not limited to this. Oxidase may be added during the amine elimination step, and further oxidase may be added in the EAP quantitative determination step.

[0135] Refer to Figure 3. First, the first oxidase is added to the sample to decompose or eliminate the amines contained in the sample (S301). Phosphatase is added to the sample and reacted with EAP to convert it to EA (S303). The second oxidase is added to the sample and reacted with the converted EA (S305). The hydrogen peroxide produced by this reaction is detected (S307). Using a calibration curve prepared in advance, EAP is quantified from the detected hydrogen peroxide value (S309).

[0136] In step S301, the oxidase used to eliminate free EA in the sample, and in step S305, the oxidase used to quantify the EA released from the target substance EAP by the action of phosphatase, may be of different types or the same type. Furthermore, the aforementioned oxidoreductase may be used to eliminate amines such as free EA in the sample. The appropriate concentrations of enzymes for elimination, quantification, and substrate specificity of the enzymes used can be appropriately determined.

[0137] Step S303 may be the same as step S101 described above. Also, step S305 may be the same as step S103 described above. Step S307 may be the same as step S105 described above. Step S309 may be the same as step S107 described above, and a detailed explanation is omitted.

[0138] <Oxidase> The oxidases usable in this invention are oxidases that act on EA as a substrate or oxidases that act on substrates structurally similar to EA. Substrates structurally similar to EA include substrates having CH-NH2 bonds or CH-NH bonds, and amine oxidases are enzymes of such substrates.

[0139] More specifically, substrates structurally similar to EA include phenylethylamine, EA, tyramine, benzylamine, histamine, serotonin, spermine, spermidine, β-alanine, γ-aminobutyric acid (GABA), taurine, cadaverine, and agmatine. Suitable oxidases for these substrates include phenylethylamine oxidase (PEAOX), ethanolamine oxidase, tyramine oxidase, benzylamine oxidase, histamine oxidase, serotonin oxidase, spermine oxidase, spermidine oxidase, β-alanine oxidase, γ-aminobutyric acid (GABA) oxidase, taurine oxidase, cadaverine oxidase, and agmatine oxidase.

[0140] The reaction conditions for the oxidase used in this invention can be any conditions that act on EA and efficiently catalyze the oxidation reaction. However, enzymes generally have an optimal temperature and pH at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperature and pH are preferable. For example, the reaction conditions for PEAOX can preferably be 37°C and pH 8.5, as described later, but are not limited to these.

[0141] In the reaction process of the oxidase used in the quantitative analysis method for EAP according to the present invention, various chemical substances may be involved when the oxidase acts on EA. For example, when the oxidase of the present invention acts on EA, oxygen may be involved as an electron acceptor in the redox reaction.

[0142] The oxidase used in the EAP quantification method according to the present invention may be an oxidase produced by naturally occurring microorganisms or an oxidase produced by a transformed microorganism. From the viewpoint of efficient high-volume expression of the enzyme, using a transformed microorganism allows for efficient high-volume expression of the enzyme.

[0143] For example, the oxidase used in the EAP quantification method according to the present invention may be oxidase produced by Arthrobacter globiformis (AgPEAOX), or oxidase produced by Escherichia coli transformed with a plasmid containing the PEAOX gene derived from Arthrobacter globiformis. However, by using Escherichia coli transformed with a plasmid containing the PEAOX gene derived from Arthrobacter globiformis, the oxidase can be expressed in large quantities efficiently.

[0144] Furthermore, for example, the oxidase used in the EAP quantification method according to the present invention may be LcAOX produced by Lichtheimia corymbifera, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the LcAOX gene having the nucleotide sequence of Sequence ID No. 3 derived from Lichtheimia corymbifera. However, by using Escherichia coli transformed with a plasmid containing the LcAOX gene derived from Lichtheimia corymbifera, the oxidase can be expressed in large quantities efficiently.

[0145] Furthermore, for example, the oxidase used in the EAP quantification method according to the present invention may be LrHP produced by Lichtheimia ramosa, or a hypothetical protein produced by Escherichia coli transformed with a plasmid containing the LrHP gene having the base sequence of Sequence ID No. 4 derived from Lichtheimia ramosa. However, by using Escherichia coli transformed with a plasmid containing the LrHP gene derived from Lichtheimia ramosa, the oxidase can be expressed in large quantities efficiently.

[0146] Furthermore, for example, the oxidase used in the EAP quantification method according to the present invention may be SrAOX3925 produced by Syncephalastrum racemosum, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the SrAOX3925 gene having the base sequence of Sequence ID No. 5 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrAOX3925 gene derived from Syncephalastrum racemosum, the oxidase can be expressed in large quantities efficiently.

[0147] Furthermore, for example, the oxidase used in the EAP quantification method according to the present invention may be SrAOX3926 produced by Syncephalastrum racemosum, or amine oxidase produced by Escherichia coli transformed with a plasmid containing the SrAOX3926 gene having the base sequence of Sequence ID No. 6 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrAOX3926 gene derived from Syncephalastrum racemosum, the oxidase can be expressed in large quantities efficiently.

[0148] Furthermore, for example, the oxidase used in the EAP quantification method according to the present invention may be SrEAOX produced by Syncephalastrum racemosum, or ethanolamine oxidase produced by Escherichia coli transformed with a plasmid containing the SrEAOX gene having the base sequence of Sequence ID No. 7 derived from Syncephalastrum racemosum. However, by using Escherichia coli transformed with a plasmid containing the SrEAOX gene derived from Syncephalastrum racemosum, the oxidase can be expressed in large quantities efficiently.

[0149] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of PEAOX produced by Arthrobacter globiformis (SEQ ID NO: 9), and an oxidase having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0150] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of LcAOX produced by Lichtheimia corymbifera (SEQ ID NO: 10), and an oxidase having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0151] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of LrHP produced by Lichtheimia ramosa (SEQ ID NO: 11), and an oxidase having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted.

[0152] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of SEQ ID NO: 12.

[0153] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of SEQ ID NO: 13.

[0154] In one embodiment, the oxidase used in the EAP quantification method according to the present invention is an oxidase having high sequence identity (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of SEQ ID NO: 14.

[0155] The amino acid sequence identity of these oxidases is calculated using the same method as that used for oxidoreductases, so a detailed explanation is omitted.

[0156] (Method for preparing oxidase) The following describes a method for preparing oxidase used in the quantitative analysis method of EAP according to the present invention.

[0157] (Construction of expression plasmids) The oxidase expression plasmid used in the EAP quantification method according to the present invention is obtained by a commonly used method. For example, DNA is extracted from a microorganism that produces the oxidase according to the present invention, and a DNA library is prepared. From the prepared DNA library, the DNA fragment encoding the oxidase is identified and isolated. Using complementary primers with the isolated DNA fragment as a template, the DNA fragment is amplified by polymerase chain reaction (PCR), and the gene encoding the oxidase is cloned. The amplified DNA fragment is ligated to a vector to obtain a plasmid containing the DNA fragment encoding the oxidase.

[0158] Alternatively, a DNA fragment encoding oxidase can be chemically synthesized, and this DNA fragment can be ligated to a vector to obtain a plasmid containing the oxidase-encoding DNA fragment.

[0159] The obtained plasmid is used to transform strains of E. coli and other bacteria to obtain strains of E. coli and other bacteria that possess DNA encoding oxidase.

[0160] Yeast or filamentous fungi may be used as host cells for oxidase expression. The method is the same as that for oxidoreductase expression described above, and a detailed explanation is omitted.

[0161] The oxidase may have amino acid substitutions that enhance its reactivity to EAP. For example, PEAOX derived from Arthrobacter globiformis having the amino acid sequence of SEQ ID NO: 9 may have amino acid substitutions at the position corresponding to phenylalanine at position 105 and / or leucine at position 358.

[0162] (Enzyme recombination expression, purification) The expression and purification of oxidase may be carried out in the same manner as the expression and purification of oxidoreductase described above, and a detailed explanation is omitted.

[0163] (Measurement of oxidase activity) Any method for measuring oxidase activity is acceptable, as long as it directly or indirectly measures the product of a reaction catalyzed by oxidase. For example, if the product of a reaction catalyzed by oxidase is reacted with a reagent that reacts with the product (hereinafter referred to as the "product reaction reagent"), and the absorbent substance produced by this reaction is measured, then enzyme activity can be measured by measuring absorbance.

[0164] In one embodiment, for example, the enzyme disclosed in Japanese Patent Application Publication No. 2014-233219 may be used as the oxidase.

[0165] (Composition containing oxidase and kit for quantitative analysis of EAP) The method for quantifying EAP using phosphatase and oxidase according to the present invention may be carried out by providing a composition containing phosphatase, oxidase, and a product reaction reagent, or by combining phosphatase, oxidase, and a commercially available product reaction reagent. For example, it may be provided as a composition for quantifying EAP containing phosphatase and oxidase, or as a composition for quantifying EAP further containing phosphatase and a reagent that reacts with hydrogen peroxide generated by adding oxidase.

[0166] In one embodiment, a kit for quantifying EAP may be provided, comprising a phosphatase, an oxidase, and a reagent that reacts with hydrogen peroxide generated by adding the oxidase. For example, the kit for quantifying EAP may comprise a first reagent comprising a phosphatase and an oxidase, and a second reagent comprising a reagent that reacts with hydrogen peroxide generated by adding the oxidase. It may further comprise a third reagent comprising an oxidoreductase or oxidase that acts on at least one amine other than ethanolamine phosphate, and the kit may be configured to add the third reagent to the sample before adding the first and second reagents.

[0167] (Sensor chip and electrodes) In one embodiment, the oxidase of the present invention may be coated, adsorbed, or immobilized on the electrode. Preferably, the oxidase of the present invention is coated, adsorbed, or immobilized on the working electrode. In another embodiment, phosphatase may also be coated, adsorbed, or immobilized on the electrode together with the oxidoreductase. The electrode configuration can be the same as the configuration described for electrodes using oxidoreductase, so a detailed explanation is omitted. Furthermore, the oxidase can be immobilized on the electrode by crosslinking, coating with a dialysis membrane, encapsulation in a polymer matrix, use of a photocrosslinkable polymer, use of an electrically conductive polymer, use of an oxidation / reduction polymer, etc.

[0168] The oxidase of the present invention can be applied to various electrochemical measurement techniques by using a potentiostat or galvanostat. Examples of electrochemical measurement techniques include amperometry, potentiometry, and coulometry. For example, using amperometry, the concentration of EAP in a sample can be calculated by measuring the current generated when the hydrogen peroxide produced by the reaction of oxidase with EAP is subjected to a hydrogen peroxide electrode with a voltage of +600mV to +1000mV (vs. Ag / AgCl). For example, a calibration curve can be created by measuring the current values ​​for known EAP concentrations (0, 50, 100, 150, 200 μM) and plotting them against the EAP concentration. The EAP concentration can then be obtained from the calibration curve by measuring the current value for an unknown EAP. For example, a carbon electrode or a platinum electrode can be used as the hydrogen peroxide electrode. Alternatively, instead of a hydrogen peroxide electrode, an electrode immobilized with reductases such as peroxidase or catalase can be used. By applying -400mV to +100mV (vs. Ag / AgCl) and measuring the resulting reduction current, the amount of hydrogen peroxide can be quantified, and the EAP value can be measured.

[0169] Furthermore, to reduce the amount of solution required for measurement, printed electrodes (sensor chips) can be used. In this case, it is preferable that the electrodes are formed on a substrate made of an insulating substrate. The configuration of the sensor chip using oxidase may be the same as that of the sensor chip using oxidoreductase, and a detailed explanation is omitted.

[0170] [EAP Measurement Sensor] In one embodiment, an EAP measurement sensor using the oxidase of the present invention is provided. The sensor is an EAP measurement device using the oxidase of the present invention, and comprises a sensor chip containing the oxidase and a measurement unit. The configuration of the EAP measurement sensor using oxidase may be the same as the configuration of the EAP measurement sensor using oxidoreductase, and a detailed explanation is omitted.

[0171] As described above, the present invention provides a method for quantifying EAP, an oxidase for quantification, a composition for quantification, and a kit for quantification, which contain an oxidase, and can provide a novel method for quantifying EAP concentration, a biomarker for depression, an enzyme for quantification, a composition for quantification, a kit for quantification, and a sensor for quantification. [Examples]

[0172] Use the following reagents. Reagents: Recombinant alkaline phosphatase, highly active (Roche) 4-aminoantipyrine (4-AA) (Fujifilm Wako Pure Chemical Industries, Ltd.), taurine (Fujifilm Wako Pure Chemical Industries, Ltd.), β-alanine (Fujifilm Wako Pure Chemical Industries, Ltd.), ethanolamine phosphate (EAP) (Tokyo Chemical Industries, Ltd.), ethanolamine (EA) (Tokyo Chemical Industries, Ltd.), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) (Dojin Chemical Laboratories Co., Ltd.), horseradish peroxidase (POD) (Toyobo Co., Ltd.), and KOD-plus-Neo (Toyobo Co., Ltd.)

[0173] (Construction of LrHP expression plasmid) The expression plasmid (pKK223-3-LrHP) for Lichtheimia ramosa-derived hypothetical protein (LrHP, GenBank ID CDS02610.1) having the amino acid sequence of SEQ ID NO: 11 was prepared using the In-Fusion® HD Cloning Kit (Clontech).

[0174] Fragments of the vector (pKK223-3) were prepared by PCR using pKK223-3-CFP-T7 (see publication WO2007 / 125779) as a template and pKK223-3 HindIII 3Fw (5'-AAGCTTGGCT GTTTTGGCGG ATGAGAGAAG-3') and pKK223-3 EcoRI 5Rv (5'-GAATTCTGTT TCCTGTGTGA AATTGTTATC-3') as primers. After PCR, 1.0 μl of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour. The solution was then subjected to agarose gel electrophoresis, and the gel containing the target fragment (approximately 4.6 kbp) was excised and extracted from the gel using the illustra® GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).

[0175] The LrHP gene containing the base sequence of Sequence ID No. 4 was split into three parts: an anterior portion (lrhp_frag1) in which the DNA sequences of Sequence ID No. 15 and No. 16 were sequentially joined from the 5' end to the 3' end; an anteroposterior portion (lrhp_frag2) described in Sequence ID No. 17; and a posterior portion (lrhp_frag3) in which the DNA sequences of Sequence ID No. 18 and No. 19 were sequentially joined from the 5' end to the 3' end; and synthesis was commissioned to Integrated DNA Technologies. To utilize an in-fusion reaction, the 3' end of lrhp_frag1 and the 5' end of lrhp_frag2 (15 bases) (CATTTAGGGCAAGAT), and the 3' end of lrhp_frag2 and the 5' end of lrhp_frag3 (15 bases) (CACCATCAACATTTG) were duplicated.

[0176] An in-fusion reaction (50°C, 15 minutes) was performed using the pKK223-3 vector fragment and three LrHP gene fragments according to the composition shown in Table 1 to obtain an LrHP expression plasmid (pKK223-3-LrHP). E. coli JM109 strain was transformed with the obtained plasmid.

[0177] [Table 1]

[0178] (Enzyme recombinant expression) LrHP-producing strains were inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) in a test tube and cultured overnight at 37°C and 160 rpm. 1.5 ml of the seed culture solution was inoculated into 150 ml of LB-amp medium (ampicillin concentration 50 μg / ml) containing 0.02 mM CuSO4 and 0.1 mM IPTG in a Sakaguchi flask and cultured at 25°C for 20 hours.

[0179] The pellet obtained by centrifuging 150 ml of culture medium at 6,500 × g for 10 minutes was resuspended in 20 mM Tris-HCl pH 7.5. After sonication of the bacterial suspension, it was centrifuged at 20,400 × g for 15 minutes, and the supernatant was collected to obtain the crude enzyme solution.

[0180] (Purification of LrHP) The crude LrHP enzyme solution was applied to HiScreen® Capto Q (manufactured by GE Healthcare, resin volume 4.7 ml) equilibrated with 20 mM Tris-HCl pH 7.5 and bound to the anion exchange resin.

[0181] Subsequently, the resin was washed with 47 ml (10 CV) of 20 mM Tris-HCl (pH 7.5), and 117.5 ml (25 CV) of the 20 mM Tris-HCl (pH 7.5) was delivered while linearly increasing the NaCl concentration from 0 mM to 500 mM, thereby eluting the LrHP bound to the resin.

[0182] The eluted fraction was diluted three times with deionized water to reduce the salt concentration, and then applied to HiScreen® Capto Q ImpRes (manufactured by GE Healthcare, resin volume 4.7 ml) equilibrated with 20 mM Tris-HCl pH 7.5 to bind to the anion exchange resin.

[0183] Subsequently, the resin was washed with 23.5 ml (5 CV) of 20 mM Tris-HCl (pH 7.5), and 141 ml (30 CV) of the 20 mM Tris-HCl (pH 7.5) was delivered while linearly increasing the NaCl concentration from 0 mM to 300 mM, thereby eluting the LrHP bound to the resin.

[0184] The eluted fraction was concentrated using Amicon Ultra Ultracel-30K and purified using a HiLoad® 26 / 60 Superdex 200 column. 10 mM Bis-Tris-HCl (pH 7.0) containing 150 mM NaCl was used for resin equilibration and elution.

[0185] The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected and used as purified LrHP standards.

[0186] An activity assay reagent with the following composition was prepared. [Table 2]

[0187] (Verification of the conversion reaction from EAP to EA by ALP) Using a sample containing 1 mM EA, the reaction reagents (pH 8.0) shown in Table 2 were mixed in a 1.5 ml microcentrifuge tube at 4°C. 2 μl of ALP (to a final concentration of 5 U / ml) was added, and the mixture was incubated at 37°C for 5 minutes. Next, 20 μl of LrHP (to a final concentration of 25 μg / ml) was added, mixed by pipetting, and transferred to a cuvette. The cuvette was immediately placed in a spectrophotometer (U-3900, Hitachi High-Tech Science Corporation), and the absorbance at 555 nm was measured over 300 seconds.

[0188] Similarly, using a sample containing 1 mMEEAP, 2 μl of alkaline phosphatase (ALP) or water (to a final concentration of 5 U / ml) was added to the reaction reagent (pH 8.0) and incubated at 37°C for 5 minutes. Then, 20 μl of LrHP (to a final concentration of 25 μg / ml) was added, mixed by pipetting, transferred to a cuvette, and immediately set in a spectrophotometer. The absorbance at 555 nm was measured over time for 300 seconds.

[0189] Figure 6 shows the measurement results of EAP oxidation activity upon ALP addition. The measurement start time is defined as 0 seconds immediately after LrHP addition. In samples containing 1 mM EAP, no change in absorbance was observed without ALP addition (indicated by black circles in the figure). On the other hand, when ALP was added, a time-dependent increase in absorbance was observed immediately after LrHP addition (indicated by black triangles in the figure). This linear increase in absorbance was similar to the increase observed when EA was used directly as a substrate instead of acting EAP with phosphatase. Therefore, it was revealed that 1 mM EAP is largely converted to EA by ALP, allowing for the measurement of absorbance changes.

[0190] (Preparation of plasma model solution) A 45 μM EAP solution containing 312 μM EA, 870 μM β-alanine, and 1185 μM taurine as interfering amines was prepared. EA, β-alanine, and taurine are amines present in relatively high concentrations in plasma. The concentration of EA was determined based on the plasma concentrations listed in SRL General Testing Guide (https: / / test-guide-en.srl.info / hachioji_en / test / detail / 00080A40H), and the concentrations of β-alanine and taurine were determined based on the plasma concentrations listed in MedlinePlus® (https: / / medlineplus.gov / ency / article / 003361.htm). The final concentration in a 600 μl measurement system was determined to be the same as the plasma concentration.

[0191] (Measurement of EAP by combining ALP and LrHP) The reaction reagents (pH 8.0) shown in Table 2 were mixed in a 1.5 ml microcentrifuge tube at 4°C and incubated at 37°C for 2 minutes. At this time, 47 μl of LrHP was added (to a final concentration of 1 mg / ml). Next, 20 μl of a 45 μM EAP solution containing EA, β-alanine, and taurine was added, mixed by pipetting, and transferred to a cuvette. The cuvette was immediately set up in a spectrophotometer, and the absorbance at 555 nm was measured for 500 seconds. The final concentrations of EA, β-alanine, taurine, and EAP were 10.4 μM, 29 μM, 39.5 μM, and 1.5 μM, respectively. The measurement was paused, 10 μl (25 U / ml) of alkaline phosphatase was added to the cuvette, mixed by pipetting, and the cuvette was immediately set up in a spectrophotometer. The absorbance at 555 nm was measured over time for 500 seconds.

[0192] Figure 7(a) shows the measurement results for a sample to which 10 μl of water was added instead of ALP, and Figure 7(b) shows the measurement results for a sample to which 10 μl of ALP was added. When LrHP was added to a sample containing EA, EAP, β-alanine, and taurine, the reaction reached equilibrium after 500 seconds, and all of the EA in the sample was reacted. When ALP was added after 500 seconds, the EAP in the sample was converted to EA, which reacted with LrHP, causing a change in absorbance. No change in absorbance occurred when ALP was not added. Therefore, it was found that when LrHP and then ALP are added in that order, EA is eliminated by LrHP, and only EAP can be measured (final EAP concentration 1.5 μM). At this time, it was found that β-alanine and taurine, which are interfering amines that are abundant in blood, do not affect the measurement of EAP.

[0193] (Quantitative analysis of EAP using ALP and LrHP) The reaction reagents (pH 8.0) shown in Table 2 were mixed in 1.5 ml of a microcentrifuge tube at 4°C and incubated at 37°C for 2 minutes. At this time, 47 μl of LrHP was added (to a final concentration of 1 mg / ml). Four types of EAP solutions containing EA, β-alanine, and taurine were prepared (EAP concentrations of 30 μM, 45 μM, 90 μM, and 150 μM). 20 μl of each EAP solution was mixed with 580 μl of the reaction reagents (pH 8.0) shown in Table 2, incubated at 37°C for 2 minutes, mixed by pipetting, transferred to a cuvette, and immediately set in a spectrophotometer. The absorbance at 555 nm was measured over time for 500 seconds. The measurement was paused, 10 μl of ALP (final concentration 25 U / ml) was added to the cuvette and mixed by pipetting. The cuvette was immediately set in a spectrophotometer, and the absorbance at 555 nm was measured over time for 500 seconds.

[0194] Figure 8 shows the results of measuring EAP when the final EAP concentration in the sample was varied to 1.0 μM, 1.5 μM, 3.0 μM, and 5.0 μM. A linear relationship was observed between the EAP concentration and the change in absorbance (R 2 (=0.9972), this elimination system demonstrated that LrHP can measure EAP at final concentrations of 1.0 μM to 5.0 μM.

[0195] (Measurement of ALP and EAP, which is a combination of EA and oxidoreductase exhibiting dehydrogenase activity) Next, we will describe an example of measuring EAP by combining AgPEAOX, which exhibits dehydrogenase activity in EA, with ALP.

[0196] In addition to the reagents mentioned above, the following reagents were used. Reagents: Alkaline phosphatase (ALP) derived from E. coli C75 (TaKaRa), 2,6-dichloroindophenol sodium salt hydrate (DCIP) (Sigma-Aldrich), and 1-Methoxy-5-methylphenazinium methylsulfate (mPMS) (Dojin Chemical Laboratories Co., Ltd.)

[0197] (Construction of AgPEAOX expression plasmid) The expression plasmid (pKK223-3-AgPEAOX) for Arthrobacter globiformis-derived phenylethylamine oxidase (AgPEAOX, UniProt ID P46881) having the amino acid sequence of SEQ ID NO: 9 was prepared using the In-Fusion® HD Cloning Kit (Clontech).

[0198] Fragments of the vector (pKK223-3) were prepared by PCR using pKK223-3-CFP-T7 (see publication WO2007 / 125779) as a template and pKK223-3 HindIII 3Fw (5'-AAGCTTGGCT GTTTTGGCGG ATGAGAGAAG-3') and pKK223-3 EcoRI 5Rv (5'-GAATTCTGTT TCCTGTGTGA AATTGTTATC-3') as primers.

[0199] After PCR, 1.0 μl of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour. The solution was then subjected to agarose gel electrophoresis, and the gel containing the target fragment (approximately 4.6 kbp) was excised. The fragment was then extracted from the gel using the illustra® GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).

[0200] The AgPEAOX gene, which has the base sequence of SEQ ID NO: 20, was split into two parts: an anterior portion (agpeaox_1-325) in which the DNA sequences of SEQ ID NO: 15 and SEQ ID NO: 21 are joined sequentially from the 5' end to the 3' end, and a posterior portion (agpeaox_321-638) in which the DNA sequences of SEQ ID NO: 22 and SEQ ID NO: 19 are joined sequentially from the 5' end to the 3' end. Synthesis of these two parts was commissioned to Integrated DNA Technologies. The 15 bases at the 5' end of agpeaox_1-325 (SEQ ID NO: 15: CAGGAAACAGAATTC) and the 15 bases at the 3' end of agpeaox_321-638 (SEQ ID NO: 19: AAGCTTGGCTGTTTT) represent sequences derived from the pKK223-3 vector. The 15 base pairs at the 3' end of agpeaox_1-325 (ATCACGTACCTGTCC) and the 15 base pairs at the 5' end of agpeaox_321-638 (ATCACGTACCTGTCC) represent sequences that overlap between the first and second halves of the AgPEAOX gene.

[0201] An in-fusion reaction (50°C, 15 minutes) was performed using the pKK223-3 vector fragment and two AgPEAOX gene fragments according to the composition shown in Table 3 to obtain an AgPEAOX expression plasmid (pKK223-3-AgPEAOX). E. coli JM109 strain was transformed with the obtained plasmid.

[0202] [Table 3]

[0203] (Recombinant expression of AgPEAOX) AgPEAOX-producing strains were inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) in a test tube and cultured overnight at 37°C and 160 rpm. 1 ml of the seed culture solution was inoculated into 150 ml of LB-amp medium (ampicillin concentration 50 μg / ml) containing 0.1 mM CuSO4 and 0.1 mM IPTG in a Sakaguchi flask and cultured at 25°C for 16 hours.

[0204] The culture solution from six Sakaguchi flasks was centrifuged at 6,500 × g for 10 minutes to obtain a pellet, which was then resuspended in 20 mM Tris-HCl pH 8.0 containing 2 mM CuSO4.

[0205] After sonication of the bacterial cell suspension, the supernatant obtained by centrifugation at 20,400 × g for 15 minutes was buffered with 20 mM Tris-HCl pH 8.0 using Amicon® Ultra Ultracel-30K (Millipore) to obtain the crude AgPEAOX enzyme solution.

[0206] (Purification of AgPEAOX) The crude AgPEAOX enzyme solution was applied to a HiScreen® Capto Q (manufactured by GE Healthcare, resin volume 4.7 ml) equilibrated with 20 mM Tris-HCl pH 8.0 and bound to the anion exchange resin.

[0207] Subsequently, the resin was washed with 20 mM Tris-HCl (pH 8.0) containing 47 ml (10 CV) of 150 mM NaCl. 164.5 ml (35 CV) of the 20 mM Tris-HCl (pH 8.0) was then delivered while linearly increasing the NaCl concentration from 150 mM to 500 mM, thereby eluting the AgPEAOX bound to the resin.

[0208] The eluted fraction was concentrated using Amicon Ultra Ultracel-30K and purified using a HiLoad® 26 / 60 Superdex 200 column. 20 mM Tris-HCl (pH 8.0) was used for resin equilibration and elution.

[0209] The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected and used as purified AgPEAOX standards.

[0210] An activity assay reagent with the following composition was prepared. [Table 4]

[0211] (Quantitative analysis of EAP using ALP and AgPEAOX) The reaction reagents (pH 8.5) shown in Table 4 were mixed in 1.5 ml of a microcentrifuge tube at 4°C and incubated at 37°C for 2 minutes. At this time, 28.2 μl of AgPEAOX was added (to a final concentration of 1 mg / ml). Three types of EAP solutions containing EA, β-alanine, and taurine (EAP concentrations of 6 mM, 15 mM, and 30 mM) were prepared. 1160 μl of the reaction reagents (pH 8.5) shown in Table 3, incubated at 37°C for 2 minutes, was added to 40 μl of each EAP solution, mixed by pipetting, transferred to a cuvette, and immediately placed in a spectrophotometer. The absorbance at 520 nm was measured over time for 500 seconds. The measurement was paused, 7.48 μl of ALP (final concentration 3.12 U / ml) was added to the cuvette and mixed by pipetting. The cuvette was immediately placed in a spectrophotometer, and the absorbance at 520 nm was measured over time for 500 seconds.

[0212] Figure 9 shows the results of measuring EAP when the final EAP concentration in the sample was changed to 0.2 mM, 0.5 mM, and 1.0 mM. A linear relationship was observed between the EAP concentration and the absolute value of the absorbance change (R 2 (=0.9954), this elimination system demonstrated that AgPEAOX can measure EAP at final concentrations of 0.2 mM to 1.0 mM. [Explanation of symbols]

[0213] 1 Working electrode, 3 Counter electrode, 5 Reference electrode, 7 Wiring section, 9 Terminal, 10 Sensor chip, 11 Substrate, 13 Spacer, 15 Cover, 19 Reaction layer, 30 Measurement section, 31 Switch, 33 Display, 100 Sensor, 110 Control section, 120 Display section, 130 Input section, 140 Memory section, 150 Communication section, 160 Power supply, 190 Wiring

Claims

1. A first oxidoreductase is added to the sample to decompose or eliminate at least one amine other than ethanolamine phosphate contained in the sample, where the ethanolamine phosphate is not eliminated, and only at least one amine other than ethanolamine phosphate is decomposed or eliminated. The condition concentration of the first oxidoreductase is set accordingly. The aforementioned sample is treated with phosphatase to convert the ethanolamine phosphate contained in the sample into ethanolamine. This involves reacting the ethanolamine with a second oxidoreductase of the same type as the first oxidoreductase, A method for quantifying ethanolamine phosphate, wherein the first oxidoreductase and the second oxidoreductase are selected from primary amine dehydrogenase, monoamine dehydrogenase, diamine dehydrogenase, polyamine dehydrogenase, ethanolamine dehydrogenase, tyramine dehydrogenase, phenylethylamine dehydrogenase, benzylamine dehydrogenase, histamine dehydrogenase, serotonin dehydrogenase, spermine dehydrogenase, spermidine dehydrogenase, β-alanine dehydrogenase, γ-aminobutyric acid (GABA) dehydrogenase, taurine dehydrogenase, cadaverine dehydrogenase, and agmatine dehydrogenase.

2. A method for determining ethanolamine phosphate according to claim 1, comprising reducing the mediator by reacting the ethanolamine with the second oxidoreductase, and determining the concentration of the ethanolamine phosphate by quantifying the reduced mediator.

3. The first oxidoreductase and the second oxidoreductase are oxidases, The oxidase is selected from primary amine oxidase, monoamine oxidase, diamine oxidase, polyamine oxidase, ethanolamine oxidase, tyramine oxidase, phenylethylamine oxidase, benzylamine oxidase, histamine oxidase, serotonin oxidase, spermine oxidase, spermidine oxidase, β-alanine oxidase, γ-aminobutyric acid (GABA) oxidase, taurine oxidase, cadaverine oxidase, and agmatine oxidase. A method for determining the concentration of ethanolamine phosphate according to claim 1, wherein the concentration of ethanolamine phosphate is determined by reacting the ethanolamine with the oxidase and quantifying the hydrogen peroxide produced or the oxygen consumed.

4. The first oxidoreductase and the second oxidoreductase are An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or A method for quantifying ethanolamine phosphate according to claim 1, comprising selecting an oxidoreductase that has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, decomposes or eliminates at least one amine, and acts on the ethanolamine.

5. The first oxidoreductase and the second oxidoreductase are oxidases, The oxidase has 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 8, decomposes or eliminates at least one amine, and acts on the ethanolamine, or An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, A method for quantifying ethanolamine phosphate according to claim 3, comprising selecting an oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, which decomposes or eliminates at least one amine and acts on the ethanolamine.

6. The first oxidoreductase and the second oxidoreductase are oxidases, The method for quantifying ethanolamine phosphate according to claim 1, wherein the oxidase is a hypothetical protein derived from Lichtheimia ramosa having the amino acid sequence of SEQ ID NO: 11, or a phenylethylamine oxidase derived from Arthrobacter globiformis having the amino acid sequence of SEQ ID NO:

9.

7. Phosphatase and, A first oxidoreductase set to a concentration that decomposes or eliminates at least one amine other than ethanolamine phosphate, leaving ethanolamine phosphate undecomposed, and decomposing or eliminating only the amines other than ethanolamine phosphate, It contains a second oxidoreductase of the same type as the first oxidoreductase that acts on ethanolamine, A composition for quantifying ethanolamine phosphate, wherein the first oxidoreductase and the second oxidoreductase are selected from primary amine dehydrogenase, monoamine dehydrogenase, diamine dehydrogenase, polyamine dehydrogenase, ethanolamine dehydrogenase, tyramine dehydrogenase, phenylethylamine dehydrogenase, benzylamine dehydrogenase, histamine dehydrogenase, serotonin dehydrogenase, spermine dehydrogenase, spermidine dehydrogenase, β-alanine dehydrogenase, γ-aminobutyric acid (GABA) dehydrogenase, taurine dehydrogenase, cadaverine dehydrogenase, and agmatine dehydrogenase.

8. The first oxidoreductase and the second oxidoreductase are An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or A composition for quantifying ethanolamine phosphate according to claim 7, selected from an oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, which decomposes or eliminates at least one amine and acts on the ethanolamine.

9. The first oxidoreductase and the second oxidoreductase are oxidases, The oxidase has 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 8, decomposes or eliminates at least one amine, and acts on the ethanolamine, or An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, A composition for quantifying ethanolamine phosphate according to claim 7, selected from an oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, which decomposes or eliminates at least one amine and acts on the ethanolamine.

10. The first oxidoreductase and the second oxidoreductase are oxidases, The ethanolamine phosphate quantitative composition according to claim 7, wherein the oxidase is a hypothetical protein derived from Lichtheimia ramosa having the amino acid sequence of SEQ ID NO: 11, or a phenylethylamine oxidase derived from Arthrobacter globiformis having the amino acid sequence of SEQ ID NO:

9.

11. Phosphatase and, A first oxidoreductase set to a concentration that decomposes or eliminates at least one amine other than ethanolamine phosphate, leaving ethanolamine phosphate undecomposed, and decomposing or eliminating only the amines other than ethanolamine phosphate, It contains a second oxidoreductase of the same type as the first oxidoreductase that acts on ethanolamine, A kit for quantifying ethanolamine phosphate, wherein the first oxidoreductase and the second oxidoreductase are selected from primary amine dehydrogenase, monoamine dehydrogenase, diamine dehydrogenase, polyamine dehydrogenase, ethanolamine dehydrogenase, tyramine dehydrogenase, phenylethylamine dehydrogenase, benzylamine dehydrogenase, histamine dehydrogenase, serotonin dehydrogenase, spermine dehydrogenase, spermidine dehydrogenase, β-alanine dehydrogenase, γ-aminobutyric acid (GABA) dehydrogenase, taurine dehydrogenase, cadaverine dehydrogenase, and agmatine dehydrogenase.

12. The first oxidoreductase and the second oxidoreductase are An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or An oxidoreductase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, or A kit for quantifying ethanolamine phosphate according to claim 11, comprising an oxidoreductase selected from having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, decomposing or eliminating at least one amine, and acting on the ethanolamine.

13. The first oxidoreductase and the second oxidoreductase are oxidases, The oxidase has 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 8, decomposes or eliminates at least one amine, and acts on the ethanolamine, or An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 9, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 10, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 11, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 12, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, An oxidase having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 13, which decomposes or eliminates at least one of the amines and acts on the ethanolamine, A kit for quantifying ethanolamine phosphate according to claim 11, comprising an oxidase selected from having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 14, which decomposes or eliminates at least one amine and acts on the ethanolamine.

14. The first oxidoreductase and the second oxidoreductase are oxidases, The ethanolamine phosphate quantitative determination kit according to claim 11, wherein the oxidase is a hypothetical protein derived from Lichtheimia ramosa having the amino acid sequence of SEQ ID NO: 11, or a phenylethylamine oxidase derived from Arthrobacter globiformis having the amino acid sequence of SEQ ID NO:

9.

15. Working electrode, counter electrode and reference electrode, The system comprises a reaction layer disposed on the working electrode, the counter electrode, and the reference electrode, The reaction layer comprises a phosphatase, a first oxidoreductase that decomposes or eliminates at least one amine other than ethanolamine phosphate, and a second oxidoreductase that acts on ethanolamine. The first oxidoreductase is configured such that the ethanolamine phosphate is not eliminated, and only amines other than the ethanolamine phosphate are decomposed or eliminated. A sensor chip used in the method for quantifying ethanolamine phosphate according to any one of claims 1 to 6.

16. A sensor comprising the sensor chip described in claim 15.

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