Method for measuring ergothioneine and measuring kit used therefor

Abstract

To provide a new method for measuring ergothioneine, and a measuring kit or the like used therefor.SOLUTION: The method for measuring ergothioneine in a test sample includes a process (1) for making ergothionase act on the test sample, and a process (2) for determining thiourocanic acid in a reaction product obtained in the process (1) by an anti-thiourocanic acid antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for measuring ergothioneine, a measurement kit used therefor, etc. In particular, the present invention relates to a method for measuring ergothioneine by treating a test sample that may contain ergothioneine with ergothionase and quantifying the produced thiourocanic acid with an antibody, and a measurement kit used therefor, etc. [Background technology]

[0002] L-ergothioneine (sometimes referred to as "EGT" or "ergothioneine" in this specification) is a sulfur-containing amino acid and is known to have a variety of physiological activities.

[0003] For example, it has been reported that EGT has the effect of improving the efficacy of immunotherapy using a cancer vaccine consisting of a tumor-associated antigen (TAA) and an adjuvant (see, for example, Non-Patent Document 1). It has also been reported that EGT has the effect of treating bacterial infections (see, for example, Patent Document 1).

[0004] Ergothioneine is a rare amino acid that can only be produced by certain bacteria, such as fungi like mushrooms and fungi like koji mold, and it also has excellent antioxidant properties. Humans can utilize ergothioneine ingested through these foods as an antioxidant in the body. Ingested ergothioneine crosses the blood-brain barrier and is stored in the central nervous system. Furthermore, it has been reported that blood ergothioneine concentrations are lower in patients with neurodegenerative diseases such as dementia and mild cognitive impairment compared to healthy individuals.

[0005] More specifically, ergothioneine has been suggested to be useful in suppressing various diseases, and has been shown to be particularly effective in suppressing neurodegenerative diseases. It is known that the plasma EGT level of people with neurodegenerative diseases is lower than that of healthy people, with the boundary being shown to be 0.81 μM. It has also been reported that there is a strong correlation between the EGT levels in plasma and whole blood, with the plasma EGT level being approximately 1.27% of the whole blood EGT level (see, for example, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 089878 [Non-patent literature]

[0007] [Non-Patent Document 1] S. Yoshida et al., Front. Immuno l.10:671(2019) [Non-patent document 2] Irwin K. Cheah et al.,ANTIOXIDANTS & REDOX SIGNALING Volume 26,Number 5,2017,193-206 Summary of the Invention [Problem to be solved by the invention]

[0008] Test reagents that may contain ergothioneine include, for example, body fluids such as saliva, serum, and urine. However, these contain contaminants, and when ergothioneine is present at low concentrations, such as in neurodegenerative diseases, attempts to measure ergothioneine by spectrometry or the like have the problem of insufficient quantitative sensitivity.

[0009] Since the above-mentioned ergothioneine is correlated with the incidence of disease, it is believed that if ergothioneine could be easily measured by immunological assay, it would contribute to further progress in basic research as well as to future use as an in vitro diagnostic agent. Therefore, the present inventors investigated the development of a technology for measuring ergothioneine simply, quickly, and at low cost.

[0010] In light of the above circumstances, an object of the present invention is to provide a novel method for measuring ergothioneine, and a measurement kit and the like used therefor. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the inventors have succeeded in creating a novel method for measuring ergothioneine, as shown below, and have found that the above object can be achieved by the above method, thereby completing the present invention.

[0012] That is, the present invention provides the following method for measuring ergothioneine.

[0013] [1] (1) a step of treating a test sample with ergothionase; and a step (2) of quantifying thiourocanic acid in the reaction product obtained in the step (1) using an anti-thiourocanic acid antibody; The method for measuring ergothioneine in a test sample, comprising:

[0014] [2] The method according to [1], wherein the sample is a body fluid.

[0015] [3] The method according to [1] or [2], wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

[0016] [4] The method according to any one of [1] to [3], wherein the sample is a concentrated body fluid sample.

[0017] [5] The method according to any one of [4], wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

[0018] [6] The method according to any one of [1] to [5], wherein the step (2) is carried out by ELISA measurement.

[0019] The present invention also relates to the following measurement kits:

[0020] [7] A kit for measuring ergothioneine in a test sample, comprising an anti-thiourocanic acid antibody, A kit for quantifying the concentration of thiourocanic acid produced by reacting ergothioneine in a test sample with ergothionase using the anti-thiourocanic acid antibody.

[0021] [8] The kit according to [7], which contains ergothionase.

[0022] [9] The kit according to [7] or [8], wherein the sample is a body fluid.

[0023]

[10] The kit according to [9], wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

[0024]

[11] The kit according to any one of [7] to

[10] , which is for ELISA measurement. [Effects of the Invention]

[0025] By using the ergothioneine measurement method of the present invention, measurement can be performed, for example, by ELISA measurement, etc., and ergothioneine can be measured more simply, quickly, and at lower cost than conventional measurement methods such as high performance liquid chromatography.

[0026] Furthermore, by using the measurement kit of the present invention, it becomes possible to easily carry out the above-mentioned method for measuring ergothioneine. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is an explanatory diagram showing the procedure for immunizing BALB / c mice in Example 1 of the present disclosure. [Figure 2] FIG. 2 is an explanatory diagram showing a method for measuring antibody titers in mouse serum (indirect ELISA) in Example 1 of the present disclosure. [Figure 3] 3 is a graph showing the results of indirect ELISA of antisera from mice immunized four times in Example 1 of the present disclosure, showing the results for sera from mice 1 to 6 and a non-immunized mouse. [Figure 4] FIG. 4 is an explanatory diagram showing a method for measuring the binding rate (indirect competitive ELISA) for a sample containing mouse serum and actual thiourocanic acid in Example 1 of the present disclosure. [Figure 5] FIG. 5 shows the results of evaluating the binding rate with thiourocanic acid using serum from each mouse in Example 1 of the present disclosure. [Figure 6] FIG. 6 is a graph showing the measurement results of the concentration and binding rate of anti-thiourocanic acid antiserum in specific mice in Example 1 of the present disclosure. [Figure 7] 7 is a graph showing the results of measuring the antibody titers of anti-thiourocanic acid monoclonal antibodies in Example 2 of the present disclosure. The results are shown for three anti-thiourocanic acid monoclonal antibodies, TU.1-1, TU.1-2, and TU.1-3. [Figure 8] FIG. 8 is a graph showing the measurement results of the reactivity (binding rate) of anti-thiourocanic acid monoclonal antibodies with thiourocanic acid in Example 2 of the present disclosure. [Figure 9] FIG. 9 is a graph showing a calibration curve for thiourocanic acid obtained from the average of duplicates using TU.1-1 in Example 2 of the present disclosure. [Figure 10]FIG. 10 is a graph showing the results of a test to confirm cross-reactivity with EGT (ergothioneine) using TU.1-1 in Example 2 of the present disclosure. [Figure 11] FIG. 11 is an explanatory diagram showing a procedure for immunizing BALB / c mice in Comparative Example 1 of the present disclosure. [Figure 12] FIG. 12 is an explanatory diagram showing a method for measuring antibody titers in mouse serum (indirect ELISA) in Comparative Example 1 of the present disclosure. [Figure 13] 13 is a graph showing the results of indirect ELISA of mouse antisera immunized four times with the EGT-methylbenzoic acid-cBSA antigen in Comparative Example 1 of the present disclosure. The results are shown for the sera of mice 1 to 6 and a non-immunized mouse. [Figure 14] 14 is a graph showing the results of indirect ELISA of mouse antisera immunized four times with the EGT-methylphenylacetic acid-cBSA antigen in Comparative Example 1 of the present disclosure. The results are shown for the sera of mice 1, 2, 5, 6, and a non-immunized mouse. [Figure 15] FIG. 15 is an explanatory diagram showing a method for measuring the binding rate (indirect competitive ELISA) for a sample containing mouse serum and actual ergothioneine in Comparative Example 1 of the present disclosure. [Figure 16] FIG. 16 shows the results of evaluating the binding rate with ergothioneine using serum from each mouse in Comparative Example 1 of the present disclosure. [Figure 17] FIG. 17 is a graph showing the measurement results of the concentration and binding rate of anti-ergothioneine antiserum in specific mice in Example 1 of the present disclosure. [Figure 18] FIG. 18 is a graph showing the correlation between ergothioneine concentration and an increase in absorption spectrum at 311 nm when ergothioneine was converted to thiourocanic acid using ergothionase in Comparative Example 3 of the present disclosure. [Figure 19] FIG. 19 is a graph showing the relationship between the binding rate and the EGT concentration in the case of ELISA of the present invention in Comparative Example 3 of the present disclosure. [Figure 20]FIG. 20 is a graph showing the measurement results of ergothioneine concentration when the sample contained whole blood in Example 5 of the present disclosure. [Figure 21] FIG. 21 is a graph showing the measurement results of ergothioneine concentration when the sample contained plasma in Example 5 of the present disclosure. [Figure 22] FIG. 22 is a graph showing the measurement results of ergothioneine concentration when semen was included in the sample in Example 5 of the present disclosure. [Figure 23] FIG. 23 shows the results of HPLC analysis showing that when ergothioneine is treated with ergothionase, ergothioneine disappears and is entirely converted to thiourocanic acid. [Figure 24] FIG. 24 shows that the ELISA results for a sample in which ergothioneine was converted with ergothionase or thiourocanic acid were measured by the ELISA method in Example 5, and that the ELISA results for both samples were consistent. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, embodiments of the present invention will be described in detail.

[0029] [Method for measuring ergothioneine] The measurement method of the present invention comprises: (1) a step of treating a test sample with ergothionase; and a step (2) of quantifying thiourocanic acid in the reaction product obtained in the step (1) using an anti-thiourocanic acid antibody; The method for measuring ergothioneine in a test sample comprises:

[0030] By using the ergothioneine measurement method of the present invention, for example, the amount of ergothioneine in a test sample can be measured by immunoassay. The measurement method of the present invention enables ergothioneine to be measured more simply, quickly, and at lower cost than conventional measurements using high-performance liquid chromatography, etc. Furthermore, the measurement method of the present invention enables highly sensitive detection and measurement of ergothioneine, even when, for example, ergotineine is present at a low concentration of 1 μM or less, making direct spectroscopic measurement difficult. Furthermore, the measurement method of the present invention enables highly sensitive detection and measurement of ergothioneine by immunoassay, even when, for example, the test sample contains impurities derived from urine, serum, or saliva.

[0031] The above-mentioned measurement method is believed to be effective through the following mechanism of action, but the scope of the patent is not limited to this mechanism. First, for example, by adding ergothionase to a test sample that may contain ergothioneine, ergothioneine, if present, is quantitatively converted to thiourocanic acid (or its equivalent). Then, by quantifying this thiourocanic acid with an anti-thiourocanic acid antibody, the ergothioneine contained in the test sample can be measured.

[0032] It is known that plasma EGT levels in people with neurodegenerative diseases are lower than those in healthy individuals, with the borderline reported to be 0.81 μM. For example, a whole blood EGT level of approximately 60-70 μM is estimated to be the borderline between healthy individuals and those with neurodegenerative diseases. Therefore, the development of a technology capable of quantifying EGT levels in whole blood down to at least 60-70 μM may be required. Even in such cases, the measurement method of the present invention allows for sufficient measurement across this concentration range.

[0033] In the present invention, the above-mentioned "measurement" also includes simple "detection" aimed simply at confirming or detecting the presence of ergothioneine.

[0034] A typical example of the above step (1) is a step of producing thiourocanic acid by adding ergothionase to a test sample that may contain ergothioneine, but the order of mixing the components and the preparation method are not particularly limited as long as the actual formulation involves a process in which the test sample that may contain ergothioneine and ergothionase are mixed together. For example, the above step (1) also includes a case in which the test sample that may contain ergothioneine is added to a solution containing ergothionase.

[0035] The ergothioneine may be L-ergothioneine, D-ergothioneine, or a mixture thereof.

[0036] The ergothioneine can be a commercially available product, one produced by a known synthesis method, or one produced by a new synthesis method, without any particular limitation.

[0037] The ergothionase is an enzyme that decomposes ergothioneine into thiourocanic acid and trimethylamine. Microorganisms having ergothionase activity include, for example, those derived from microorganisms belonging to the genus Burkholderia (Burkholderia sp.) and the genus Pseudomonas (Pseudomonas sp.), but the origin is not limited thereto. Other commercially available, known, or novel ergothionases can also be used, and are not particularly limited as long as they have the ability to decompose ergothioneine into thiourocanic acid and trimethylamine. These may be used alone or in combination of two or more.

[0038] The step (1) can be carried out, for example, at a pH of 5 to 10.0, preferably 6 to 9.5, or more preferably 7 to 9.0. This can be appropriately set depending on the chemical reaction and the enzyme activity. Furthermore, although the treatment is usually carried out in an environment of around 9.0, which is near the optimal pH of the enzyme used, in the present invention, it is preferable to carry out the treatment in a pH environment of, for example, 7.3, 7.4, or 7.5, taking into account the subsequent ELISA treatment.

[0039] The above step (1) can be carried out at a temperature ranging from 15 to 45° C., preferably from 20 to 40° C. The reaction time is not particularly limited, but can be set to about 1 minute to 24 hours, preferably about 10 to 120 minutes.

[0040] In the above step (1), the concentrations of the test sample that may contain ergothioneine and ergothionase before addition (before preparation) can be adjusted appropriately depending on the purpose and application of the use.

[0041] Here, the test sample includes, for example, a body fluid derived from a subject, and refers to not only the body fluid itself, but also a concentrated body fluid, a diluted body fluid, or other appropriately treated liquid. Here, body fluid refers to, but is not limited to, urine, blood (whole blood, plasma, serum), sputum, stool, sweat, cerebrospinal fluid, digestive fluid, ascites, sweat, bile, gastric juice, tears, nasal fluid, breast milk, semen, seminal plasma, earwax, lymph, etc. Preferably, the body fluid is, for example, urine, saliva, semen, seminal plasma, or blood (whole blood, plasma, serum).

[0042] Here, more specifically, the treatment of body fluids refers to pretreatments such as concentration, dilution, fractionation, heating, and desalting, as well as the addition of preservatives such as glycerin, stabilizers such as protease inhibitors, and antiseptics. It also includes returning the body fluid to room temperature after refrigeration or freezing, and appropriate treatments either before or after refrigeration or freezing. Furthermore, for example, when the body fluid is blood, appropriate treatments include treatment with an anticoagulant, rupturing cells by dilution with water, or denaturing and removing proteins by heat treatment or the addition of organic solvents (e.g., methanol, acetone, acetonitrile). These treatments can also be combined.

[0043] Furthermore, for example, when the body fluid is serum, plasma, urine, saliva, etc., it is preferable to carry out a procedure including concentration as a pretreatment before measurement. This concentration method is not particularly limited, but examples include a method using an ultrafiltration membrane with a molecular weight cutoff, freeze concentration, reduced pressure or vacuum concentration, heating, etc.

[0044] The dilution can be carried out using, for example, distilled water or a buffer solution, and can also be used to prepare concentrated urine.

[0045] In the above step (1), the concentration of ergothioneine in a test sample that may contain ergothioneine is, for example, 10 nM to 100 mM, or may be 50 nM to 5 mM, 100 nM to 3 mM, 200 nM to 2 mM, 300 nM to 1 mM, etc., and may be appropriately adjusted to be within the above range. Furthermore, a test sample that may contain ergothioneine may not contain ergothioneine as a result, and may be, for example, 0 to 100 mM, 0 to 5 mM, 0 to 3 mM, 0 to 2 mM, 0 to 1 mM, etc.

[0046] The above step (2) is a step of quantifying thiourocanic acid in the reaction product obtained in the above step (1) using an anti-thiourocanic acid antibody. The method for the above quantification is not particularly limited as long as it is a method that can detect and measure thiourocanic acid using an antibody.

[0047] The above step (2) can be, for example, a method of detection by immunoassay. Suitable immunoassays include, but are not limited to, ELISA, dot blot, Western blot, and radioimmunoassay. Among these, ELISA is preferred, and competitive ELISA is particularly preferred. ELISA may be either direct ELISA or indirect ELISA. As a result, ergothioneine can be measured more simply, rapidly, and at lower cost than conventional methods such as high-performance liquid chromatography.

[0048] Furthermore, for example, prior to the above step (2), an antibody capable of quantifying thiourocanic acid (anti-thiourocanic acid antibody, antibody specific to thiourocanic acid) is prepared. Here, the antibody is not particularly limited as long as it is an antibody capable of quantifying thiourocanic acid. The antibody may be produced using a known method for producing anti-thiourocanic acid antibodies using thiourocanic acid or a derivative thereof as an antigen. The antibody may be either a monoclonal antibody or a polyclonal antibody, with monoclonal antibodies being preferred. Furthermore, the antibody may be a functional fragment (antigen-binding fragment) of such a monoclonal antibody. Examples of methods for producing the antibody include the methods described in the Examples of the present disclosure.

[0049] As a more specific example of how to produce the antibody, a thiourocanic acid derivative is first prepared as an immunogen by binding thiourocanic acid to a protein (e.g., cationized bovine serum albumin (cBSA), ovalbumin (OVA), etc.) via a linker. Examples of the linker include, but are not limited to, a hydrocarbon group (e.g., a benzoic acid structure).

[0050] Next, for example, mice are immunized with the immunogen by repeated subcutaneous inoculation, blood is collected, the antibody titer is evaluated, and the target anti-thiourocanic acid antibody is confirmed and evaluated. Then, for example, spleen cells from mice bearing the anti-thiourocanic acid antibody are collected and fused with mouse myeloma cells. After HAT medium selection and screening (ELISA, etc.) are performed once or multiple times as necessary, hybridomas producing the anti-thiourocanic acid antibody are collected and mass-cultured, whereby the anti-thiourocanic acid monoclonal antibody can be produced.

[0051] More specifically, in step (2), for example, an indirect competitive ELISA technique can be used. Indirect competitive ELISA can be performed according to the following procedure.

[0052] First, a thiourocanic acid derivative in which a protein is bound to thiourocanic acid via a linker is immobilized on a carrier. A microtiter plate or the like is preferably used as the carrier. Immobilization is performed, for example, by placing a buffer solution containing the thiourocanic acid derivative for immobilization (antigen for immobilization) on the carrier and incubating it. The concentration of the antigen in the buffer solution is approximately 0.001 μg / mL to 100,000 μg / mL. Examples of buffer solutions that can be used include phosphate buffer and sodium bicarbonate buffer.

[0053] Next, the carrier surface is blocked to prevent nonspecific adsorption to the carrier. Examples of blocking substances that can be used include ovalbumin (OVA) and bovine serum albumin (BSA). Blocking is performed by adding these substances to the carrier and incubating overnight at about 4°C with, for example, Dulbecco's phosphate-buffered saline (PBS), followed by washing with a wash solution containing, for example, PBS.

[0054] A solution containing the reaction product obtained in step (1) and an anti-thiourocanic acid antibody, each containing various concentrations of thiourocanic acid, is added to the blocked solid surface, and the antibody is allowed to competitively react with the immobilized antigen and thiourocanic acid to form an immobilized antigen-antibody complex and a thiourocanic acid-antibody complex. The reaction can be carried out, for example, at room temperature for 30 minutes to several hours.

[0055] By measuring the amount of the immobilized antigen-antibody complex, the amount of thiourocanic acid in the reaction product can be determined from a previously prepared calibration curve. An enzyme-labeled secondary antibody is used to measure the amount of the immobilized antigen-antibody complex. The secondary antibody is an antibody that recognizes the thiourocanic acid antibody, and can be, but is not limited to, a goat anti-mouse IgG antibody. The enzyme label can be, for example, peroxidase or alkaline phosphatase. The reaction can be carried out, for example, at room temperature for 30 minutes to several hours.

[0056] The amount of thiourocanic acid can be calculated from a calibration curve by adding a chromogenic substrate that reacts with the labeling enzyme of the secondary antibody and measuring the absorbance. The chromogenic substrate can be a chromogenic substrate solution containing a substrate appropriate for the type of enzyme, such as, but not limited to, hydrogen peroxide, 3,3',5,5'-tetramethylbenzidine, or o-phenylenediamine. After stopping the enzyme reaction, the concentration of thiourocanic acid can be detected by measuring the absorbance.

[0057] For example, the inhibition rate is calculated as the rate of decrease in absorbance of a solution containing thiourocanic acid and reacted with an antibody relative to the absorbance of a reaction solution containing no thiourocanic acid. The concentration of thiourocanic acid in the reaction product can be calculated using a calibration curve prepared in advance based on the inhibition rates of a reaction solution containing a known concentration of ergothioneine converted to thiourocanic acid by ergothionase or a reaction solution containing a known concentration of thiourocanic acid.

[0058] In the above step (2), for example, when absorbance measurement is used, it can be performed by measuring absorbance at, for example, 300 to 550 nm. The wavelength for the absorbance measurement is appropriately determined depending on the enzyme and substrate used. For example, the range can be set such that the lower and upper limits are any two points selected from the following: 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, and 550 nm.

[0059] Furthermore, the above measurement method may include, for example, after the step (2), a step (3) of evaluating the ergothioneine concentration in the test sample based on the quantitative value obtained in the step (2).

[0060] The step of evaluating the ergothioneine concentration in the test sample in step (3) above is not particularly limited, and known methods can be used. For example, when ergothioneine is present, the ergothioneine is quantitatively converted to thiourocanic acid as described in Examples 23 and 24. Therefore, a method can be used in which the molar concentration of thiourocanic acid quantified in step (2) above is evaluated as the molar concentration of ergothioneine.

[0061] More specifically, for example, as shown in Example 4, the amount of thiourocanic acid quantified by measuring the increase in absorption at 311 nm can be evaluated as the ergothioneine concentration (FIG. 18).

[0062] In the above measurement method, a known preparation step, adjustment step, post-treatment step, or the like may be provided prior to, after, or both of the above steps depending on the purpose and application.

[0063] The above-described measurement method has sufficient detection sensitivity by immunological measurement, enabling measurement of ergothioneine, even when the test sample contains impurities, for example.

[0064] The above-described measurement method can also be used, for example, for testing for various diseases associated with ergothioneine (e.g., neurodegenerative diseases such as dementia and mild cognitive impairment). As described above, ingested ergothioneine crosses the blood-brain barrier and accumulates in the central nervous system. However, it is known that in cases of neurodegenerative diseases such as dementia and mild cognitive impairment, the amount of accumulated ergothioneine is reduced compared to healthy individuals. Therefore, in one embodiment, the measurement method of the present invention may be used to detect a decrease in the amount of ergothioneine in a test sample or to determine the risk of a disease or condition associated with a decrease in ergothioneine. In another embodiment, the test sample may be derived from a subject predicted to have a decrease in ergothioneine. The subject may be, for example, an elderly person, a person suffering from a neurodegenerative disease such as dementia or mild cognitive impairment, or a person at risk of such a disease, or a person concerned about future dementia or mild cognitive impairment. In addition to the above, examples of neurodegenerative diseases include Parkinson's disease, Huntington's disease, ALS (amyotrophic lateral sclerosis), dementia with Lewy bodies, frontotemporal dementia, prion disease, and multiple sclerosis.

[0065] [Measurement kit] The measurement kit of the present invention comprises: A kit for measuring ergothioneine in a test sample, comprising an anti-thiourocanic acid antibody, The kit is characterized in that the concentration of thiourocanic acid produced by reacting ergothioneine in a test sample with ergothionase is quantified using the anti-thiourocanic acid antibody.

[0066] The above-mentioned measurement kit may preferably contain ergothionase.

[0067] In the above-mentioned measuring kit, each component is the same as in the above-mentioned measuring method.

[0068] Furthermore, by using the measurement kit of the present invention, it becomes possible to easily carry out the above-mentioned method for measuring ergothioneine. The above-mentioned kit can be suitably used, for example, in the above-mentioned method for measuring ergothioneine.

[0069] The anti-thiourocanic acid antibody is not particularly limited as long as it is an antibody that specifically recognizes thiourocanic acid, and examples thereof include the anti-thiourocanic acid antibodies used in the above-mentioned measurement methods.

[0070] The measurement kit may also contain other reagents, such as diluents for test samples, buffers, etc. The reagents may be provided, for example, in suitable sealed containers.

[0071] Furthermore, examples of test tools that may be included in the measurement kit include, but are not limited to, known disposable tools used for sampling from test samples and microtubes for mixing each reagent.

[0072] The kit may also include an instruction manual describing protocols and the like required for using the kit. The instruction manual may be printed or may be recorded as data on a known recording medium.

[0073] The above-mentioned measurement kit can be suitably used for ELISA measurement, for example.

[0074] In the above measurement kit, the test sample may be, for example, a body fluid. For example, the kit can be used when the test sample is blood (serum, etc.), urine, saliva, stool, cerebrospinal fluid, etc. Furthermore, the above measurement kit can be used when the test sample is concentrated serum, urine, saliva, stool, cerebrospinal fluid, or other body fluids.

[0075] The above-described measurement kit can also be used for testing for various ergothioneine-related diseases (e.g., neurodegenerative diseases such as dementia and mild cognitive impairment). As described in the measurement method section above, in one embodiment, the measurement kit of the present invention may be used to detect a decrease in ergothioneine levels in a test sample or to determine the risk of a disease or condition associated with a decrease in ergothioneine levels. In another embodiment, the test sample may be derived from a subject predicted to have a decrease in ergothioneine levels. The subject may be, for example, an elderly person suffering from a neurodegenerative disease such as dementia or mild cognitive impairment, or at risk of such a disease, or a person concerned about future dementia or mild cognitive impairment. In addition to the above, examples of neurodegenerative diseases include Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies, frontotemporal dementia, prion disease, and multiple sclerosis. [Example]

[0076] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0077] Example 1 Thiourocanoic acid (hereinafter also referred to as "TU") is produced, for example, by treating ergothioneine with ergothionase, as shown below, and has a structure in which the trimethylamino group has been removed from ergothioneine.

[0078] [ka]

[0079] First, the following methylbenzoic acid derivative of thiourocanoic acid and butyric acid derivative of thiourocanoic acid were prepared by a method similar to that described in JP 2023-180566. These thiourocanoic acid derivatives can be conjugated with cBSA or OVA by a commonly used protein conjugation method using water-soluble carbodiimide. Note that cBSA is cationized bovine serum albumin, and OVA is ovalbumin.

[0080] [ka]

[0081] Six BALB / c mice (Japan SLC Co., Ltd.) were immunized four times with the prepared immunogen. The immunization procedure was as shown in Figure 1. Antibody titers were measured by indirect ELISA using serum collected after immunization. The indirect ELISA was performed under the conditions shown in Figure 2.

[0082] Figure 3 shows the results of indirect ELISA of mouse antisera immunized four times. More specifically, we measured the antibody titer of mouse antisera immunized four times with thiourocanic acid-methylbenzoic acid-cBSA. The results showed that the antibody titer was higher than that of non-immunized mouse antisera, and the antibody titer of Mouse 2 antisera was particularly high. These results suggest that TU-specific antibodies may have been induced.

[0083] Next, an indirect competitive ELISA was performed to confirm the binding ability of the antiserum to TU. The indirect competitive ELISA was performed under the conditions shown in Figure 4. In addition, before adding the mouse antiserum to each well, a TU preparation was added to perform an inhibition reaction. Other than this procedure, the procedure was the same as the indirect ELISA described above.

[0084] Figure 5 shows the results of the indirect competitive ELISA. More specifically, the binding of TU to the solid-phase antigen TU-butyric acid-OVA was confirmed. This indirect competitive ELISA confirmed that the binding rate of mouse antisera decreased in a concentration-dependent manner with TU, except for mouse 6. These results confirmed the presence of TU-specific antibodies in the mouse antisera. Figure 6 also shows the relationship between the concentration of anti-TU antisera and the binding rate. The indirect competitive ELISA showed that mouse 4 antisera, the source of cell fusion, showed 20% inhibition at 8 μg / mL, resulting in a detection sensitivity of 8 μg / mL. Furthermore, these results indicated that detection was possible at lower concentrations than when the immunogen and solid-phase antigen were prepared by substituting ergothioneine with thiourocanic acid and binding to cBSA and OVA, respectively, in the same manner as above.

[0085] Example 2 Next, anti-thiourocanic acid monoclonal antibodies were produced by immunizing mice using the methylbenzoic acid derivative of thiourocanic acid in Example 1 above as an immunogen to produce anti-thiourocanic acid monoclonal antibodies.

[0086] More specifically, spleen cells were isolated from mice after the final immunization and fused with mouse myeloma cells. After two rounds of HAT selection and ELISA screening, antibody-producing hybridomas were obtained. These hybridomas were then mass-cultured in T-75 flasks to produce anti-thiourocanic acid monoclonal antibodies. The resulting three anti-thiourocanic acid monoclonal antibodies, designated TU.1-1, TU.1-2, and TU.1-3, were evaluated. The results are shown in Figures 7 and 8. More specifically, Figure 7 shows the titer measurements of the anti-thiourocanic acid monoclonal antibodies. Figure 8 shows the reactivity (binding rate) of the anti-thiourocanic acid monoclonal antibodies with thiourocanic acid. The measurements were performed using the same indirect ELISA technique as in Example 1. The results demonstrated that all antibodies possessed excellent properties, with the antibody designated TU.1-1 exhibiting particularly excellent sensitivity. Specifically, as shown in Figures 9 and 19, using this antibody, it was possible to measure extremely low concentrations of TU itself and TU converted from EGT by ergothionase, approximately 300-600 nM (69-137 ng / mL in terms of EGT).

[0087] Example 3 Next, the amount of thiourocanic acid was measured using the monoclonal antibody obtained in Example 2 under the conditions shown in Table 1. TU.1-1 was used as the monoclonal antibody.

[0088] [Table 1]

[0089] Figure 9 shows the calibration curve for thiourocanic acid obtained from the average of two measurements using TU.1-1, and Figure 10 shows the results of a test to confirm cross-reactivity with EGT (ergothioneine) using TU.1-1. The above results confirmed that the anti-thiourocanic acid monoclonal antibody of the present invention can be used to adequately measure thiourocanic acid at low concentrations, for example, around 100 ng / mg.

[0090] Comparative Example 1 The present researchers investigated the production of antibodies using ergothioneine (hereinafter referred to as "EGT") derivatives as antigens. First, the following methylphenyl derivatives of ergothioneine, methylbenzoic acid derivatives of ergothioneine, and butyric acid derivatives of ergothioneine were produced. Note that cBSA is cationized bovine serum albumin, and OVA is ovalbumin.

[0091] [ka]

[0092] Six BALB / c mice (Japan SLC Co., Ltd.) were immunized four times with the prepared immunogen. The immunization procedure was as shown in Figure 11. Antibody titers were measured by indirect ELISA using serum collected after immunization. The indirect ELISA was performed under the conditions shown in Figure 12.

[0093] Figures 13 and 14 show the results of indirect ELISA of the mouse antisera. More specifically, the antibody titers of the antisera from mice immunized four times with each immunogen were measured. As a result, the antibody titers of mice immunized with EGT-methylbenzoic acid-cBSA were higher than those of antisera from unimmunized mice. On the other hand, the antibody titers of the serum from mice immunized with EGT-methylphenylacetic acid were not higher than those of mice immunized with EGT-methylbenzoic acid-cBSA.

[0094] Next, an indirect competitive ELISA was performed to confirm the binding ability of the antiserum from EGT-methylbenzoic acid-cBSA-immunized mice to EGT. The indirect competitive ELISA was performed under the conditions shown in Figure 15. In addition, before adding the mouse antiserum to each well, an EGT standard was added to perform an inhibition reaction. Other than this procedure, the procedure was the same as the indirect ELISA described above.

[0095] Figure 16 shows the results of indirect competitive ELISA. More specifically, binding to EGT was confirmed using the solid-phase antigen EGT-butyrate-OVA. This indirect competitive ELISA confirmed that the binding rate of mouse antisera, except for Mouse 1, decreased in a concentration-dependent manner. These results confirmed the presence of EGT-specific antibodies in the mouse antisera. Furthermore, we decided to perform cell fusion using Mouse 4, which had a high antibody titer and high EGT specificity.

[0096] Comparative Example 2 Next, an anti-ergothioneine monoclonal antibody was produced by immunizing a mouse using the methylbenzoic acid derivative of ergothioneine in Comparative Example 1 as an immunogen to produce an anti-ergothioneine monoclonal antibody.

[0097] More specifically, spleen cells were extracted from mice after the final immunization and fused with mouse myeloma cells. After two rounds of HAT selection, ELISA screening, and cloning, antibody-producing hybridomas were obtained, which were then mass-cultured in T-75 flasks to produce anti-ergothioneine monoclonal antibodies.

[0098] The three anti-ergothioneine monoclonal antibodies obtained were named EGT.1-1, EGT.1-2, EGT.1-3, and EGT.1-4, and were evaluated. The EGT.1-1 antibody, which had the highest sensitivity, was used in competitive ELISA. The results are shown in Figure 17.

[0099] More specifically, Figure 17 shows the results of measuring the reactivity (binding rate) of anti-ergothioneine monoclonal antibodies with ergothioneine. The measurement was performed using the same indirect ELISA technique as in Comparative Example 1. As a result, the sensitivity was lower than that of the mouse 4 antiserum used as the basis for cell fusion, with 20% inhibition occurring at 30 μg / mL, and the detection sensitivity was 30 μg / mL (131 μM).

[0100] Thus, when measuring ergothioneine using an ergothioneine derivative as an antigen, for example, the EGT concentration in human blood is approximately 100-500 μM in healthy individuals, but because ergothioneine is primarily present in red blood cells, a hemolysis procedure is required, and the sample must be diluted 10-fold with water. Therefore, the sample concentration when subjected to ELISA is 10-50 μM, which is inappropriate for detection in the very low concentration range (high sensitivity measurement) required for the applications of the present invention.

[0101] Example 4 An ergothionase crude enzyme solution was prepared using the same method as in Example 10 of JP 2012-65591 A. The resulting ergothionase crude enzyme solution was added to an ergothioneine preparation to a concentration of 0.1%, and the mixture was incubated at room temperature for 1 hour to convert it to thiourocanic acid. This resulted in a change in the absorption spectrum, with an increase in absorbance at 311 nm ( FIG. 18 ). This increase was proportional to the ergothioneine concentration used, indicating that EGT concentration can be quantified by measuring the increase in absorbance at 311 nm. This demonstrates that the quantitative results obtained using the anti-thiourocanic acid antibody of the present invention can be easily converted into quantitative ergothioneine concentration.

[0102] Comparative Example 3 On the other hand, when ergothioneine was converted to thiourocanic acid using ergothionase and quantified by measuring the absorbance of the thiourocanic acid (hereinafter referred to as the "ETL method"), the transmittance at OD311 nm of four whole blood samples was very low, as shown in Table 2, making some samples unmeasurable. Even for the samples that could be measured, there was a large discrepancy compared with the HPLC results, indicating very low quantitation accuracy. The whole blood samples were prepared using the same method as for ELISA. That is, whole blood was diluted 10-fold with water and hemolyzed, then heat-denatured, and the centrifuged supernatant was converted with ergothionase and used for measurement. This method proved difficult to use for samples with high absorbance around 311 nm.

[0103] [Table 2]

[0104] Comparative Example 4 When the measurement value (absorbance) increases depending on the concentration of the substance being measured, as in the ETL method, the method of calculating the lower limit of quantitation is generally to measure the noise level (basal absorbance) and use 10 times the standard deviation of that basal as the lower limit of quantitation.

[0105] Therefore, the absorbance at 311 nm of 200 μL / well was measured at six points in the basal state (when only ETL was added to the buffer) without the substance to be measured, and the absorbance value calculated from 10 times the standard deviation was used as the lower limit of quantification for the ETL method.The EGT concentration at this value was calculated to be 7.2 μM (see Table 3 and Figure 18).

[0106] [Table 3]

[0107] As mentioned above, the boundary between EGT levels in whole blood of patients with neurodegenerative diseases and healthy individuals is estimated to be 60-70 μM. Because EGT in whole blood is mainly present in red blood cells, it must be hemolyzed by diluting with water or other methods. However, in order to measure EGT levels down to approximately 70 μM, the ETL method can only dilute whole blood up to 10 times.

[0108] On the other hand, in the case of competitive ELISA, the measured value (absorbance) decreases with increasing concentration of the analyte. Therefore, the lower limit of quantitation is generally determined as the concentration of the analyte at which the binding rate is 80%, assuming that the binding rate in the absence of the analyte is 100%. In this ELISA, the EGT concentration at which the binding rate is 80% was approximately 0.3-0.5 μM (see Figure 19, data obtained by converting various concentrations of EGT standard with ergothionase and applying it to ELISA).

[0109] Thus, due to the difference in the lower limit of quantitation, the ETL method required at least 14 times the sample volume. This difference is significant for precious biological samples, and the ELISA method of the present invention, which enables measurement with a smaller amount of sample, has been confirmed to be a great advantage.

[0110] Example 5 The ergothioneine concentration was measured using whole blood, plasma, and semen as samples.

[0111] More specifically, whole blood samples were prepared by placing whole blood in an Eppendorf tube and diluting it 10-fold with purified water (Direct-Q3UV-R, Millipore), followed by heating at 85°C for 8 minutes. The supernatant was obtained by centrifugation at 15,000 rpm for 15 minutes. The supernatant was then diluted appropriately with phosphate buffered saline (hereinafter referred to as PBS(-)) to prepare whole blood samples.

[0112] Plasma sample: Whole blood was collected and mixed with a small amount of heparin (Fujifilm). The mixture was centrifuged at 1500 G for 15 minutes. 100 μL of the resulting supernatant was mixed with 400 μL of methanol and incubated at -20°C for 1 hour. The supernatant was then centrifuged at 15,000 rpm for 15 minutes. The supernatant was dried using a centrifugal evaporator (CC-105, Tomy). The supernatant was dissolved in PBS (-) and used as the plasma sample.

[0113] Semen sample: After leaving the semen at room temperature for 30 minutes, 160 μL of water was added to 40 μL of semen and heated at 85°C for 8 minutes. 800 μL of methanol was added, mixed, and left at -20°C for 1 hour. The supernatant obtained after centrifugation at 15,000 rpm for 15 minutes was collected and dried in a centrifugal evaporator (CC-105: Tomy Co.). The semen was dissolved in PBS (-) and used as a plasma sample.

[0114] HPLC: An equal volume of acetonitrile was added to each sample obtained above, and the mixture was centrifuged at 15,000 rpm for 15 minutes. The supernatant was then subjected to HPLC analysis under the following conditions: HPLC system: 20AC system (Shimadzu Corporation), column: Poroshell 120 HILIC-Z (Agilent Corporation), eluent: (A) 20 mM ammonium formate pH 3.0, eluent (B): 20 mM ammonium formate / 90% acetonitrile pH 3.0, 95% B isocratic, flow rate: 0.5 mL / min, injection volume: 5 μL, detection: 263 nm.

[0115] ELISA: To an ergothioneine standard (Tetraedron) dissolved in PBS(-) and each sample dissolved in PBS(-) obtained above, the ergothioneinase crude enzyme solution obtained in Example 4 was added at a concentration of 0.05%. After incubation at 37°C for 1 hour, ELISA measurement was performed in the same manner as in the indirect ELISA in Example 1.

[0116] Regarding the experiment in Figure 23, the following experiment was conducted to confirm whether ergothioneine could be converted to thiourocanic acid by incubating it for 1 hour with 0.05% ergothionase crude enzyme solution obtained in Example 4 / PBS(-).

[0117] Ergothioneine standard and ergothioneine solution in PBS(-) were incubated with 0.05% ergothionase at 37°C for 1 hour and then subjected to HPLC. HPLC conditions were: HPLC system: 20AC system (Shimaz), column: Microbondasphere (Waters), eluent: (A) 0.1% formic acid, eluent (B) acetonitrile, gradient, flow rate: 0.5 mL / min, injection volume: 5 μL, detection: 263 nm.

[0118] Regarding the experiment in Figure 24, the following experiment was performed to confirm whether the same results would be obtained when a sample obtained by treating an ergothioneine preparation with ergothionase and a thiourocanic acid preparation were subjected to ELISA.

[0119] Ergothioneine at various concentrations dissolved in PBS(-) was added to the crude ergothionase solution obtained in Example 4 at a concentration of 0.05%, and the mixture was incubated for 1 hour at 37° C. These and a thiourocanic acid preparation dissolved in PBS(-) were measured using the same method as in the indirect ELISA described in Example 1.

[0120] The results are shown in Figures 20 to 24. As shown in Figures 20 to 24, it was found that even when the sample contained whole blood, plasma, or semen, ergothioneine could be measured by the method of the present invention in the same way as by HPLC.

[0121] Example 6 Synthesis of thiourocanoic acid in Example 1 One gram of ergothioneine (Tetraedron) was dissolved in 40 mL of 250 mM sodium carbonate buffer, pH 9.0. The crude ergothionase solution obtained in Example 4 was added to the solution to a concentration of 2%, and the mixture was incubated at room temperature for 48 hours. Hydrochloric acid was added to adjust the pH to 2.5, resulting in the formation of a precipitate. The mixture was centrifuged at 3000 rpm for 10 minutes and the precipitate was recovered. The precipitate was then added with 40 mL of 5 mM hydrochloric acid, stirred, centrifuged, and the precipitate was recovered, a process repeated twice. The precipitate was dried, and a portion was dissolved in dimethyl sulfoxide d6 (Sigma-Aldrich) and subjected to NMR (Ascend 400, BRUKER). The resulting product was confirmed to be thiourocanic acid. 1 H-NMR δ(ppm):6.311(d,1H J=15.9),7.205(d,1H J=15.9),7.359(s,1H),12.398(s,1H), 13 C-NMR δ(ppm):117.51,122.41,127.47,130.73,165.33,169.19

[0122] Example 7 NMR of the thiourocanoic acid derivative in Example 1 Each was dissolved in dimethyl sulfoxide d6 and measured by NMR (Ascend 400, BRUKER).

[0123] Thiourocanoic acid derivatives for solid-phase antigens [ka] 1 H-NMR δ(ppm):1.842(m,2H),2.306(dd,2H,J=7.4Hz),4.109(t,2H,J=6.5Hz),6.357(d,1H,J=15.8Hz),7.243(d,1H,J=15.8Hz),7.372(s,1H), 13 C-NMR δ(ppm):24.29,30.62,63.66,114.96,121.93,126.25,130.03,164.37,166.66,174.43

[0124] Thiourocanoic acid derivatives for immunogens [ka] 1 H-NMR δ(ppm):5.258(s,2H),6.432(d,1H,J=15.9Hz),7.315(d,1H,J=15.9),7.400(s,1H),7.497(d,2H,J=8.4Hz),7.953(d,2H,J=8.4Hz), 13 C-NMR δ(ppm):65.35,114.42,122.31,126.22,128.20,129.96,130.60,130.87,141.68,164.47,166.46,167.53

[0125] Example 8 Coupling of immunogenic thiourocanic acid derivatives with protein cBSA in Example 1 Ten mg of cBSA (Thermofisher) was dissolved in 2 mL of 100 mM MES buffer, pH 4.5 (Nacalai Tesque). To this solution, 5 mg of the immunogen thiourocanic acid derivative dissolved in 0.5 mL of DMSO (Fujifilm) was added dropwise with stirring. Immediately, 60 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (Tokyo Chemical Industry Co., Ltd.) was added and the mixture was stirred at room temperature for 18 hours. After removing the precipitate by centrifugation, the supernatant was loaded into a dialysis membrane (Slide A: Thermofisher) and dialyzed against 1 L of PBS(-) at 4°C for one week, changing the PBS(-) every two days. The material remaining in the dialysis membrane was used as the immunogen.

[0126] Example 9 Coupling of thiourocanoic acid derivatives for solid-phase antigens with protein OVA in Example 1 10 mg of OVA (Merck) was dissolved in 2 mL of 100 mM MES buffer, pH 4.5 (Nacalai Tesque). A solution of 5 mg of thiourocanic acid derivative (for solid-phase antigen) in 0.5 mL of DMSO (Fujifilm) was added dropwise to the solution while stirring. Immediately, 60 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (Tokyo Chemical Industry Co., Ltd.) was added and stirred at room temperature for 18 hours. After removing the precipitate by centrifugation, the supernatant was loaded into a dialysis membrane (Slide A: Thermofisher) and dialyzed against 1 L of PBS(-) at 4°C for one week, exchanging the PBS(-) every two days. The material remaining in the dialysis membrane was used as the solid-phase antigen.

Claims

1. (1) a step of treating a test sample with ergothionase; and (2) quantifying the amount of thiourocanic acid in the reaction product obtained in the step (1) using an anti-thiourocanic acid antibody; The method for measuring ergothioneine in a test sample, comprising:

2. The method of claim 1 , wherein the sample is a body fluid.

3. 10. The method of claim 1, wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

4. The method of claim 1 , wherein the sample is a bodily fluid-enriched sample.

5. 5. The method of claim 4, wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

6. The method according to any one of claims 1 to 5, wherein step (2) is carried out by ELISA measurement.

7. A kit for measuring ergothioneine in a test sample, comprising an anti-thiourocanic acid antibody, A kit for quantifying the concentration of thiourocanic acid produced by reacting ergothioneine in a test sample with ergothionase using the anti-thiourocanic acid antibody.

8. The kit of claim 7, comprising ergothionase.

9. The kit of claim 7 , wherein the sample is a body fluid.

10. 10. The kit of claim 9, wherein the sample is blood, serum, plasma, semen, seminal plasma, urine, saliva, stool, or cerebrospinal fluid.

11. The kit according to any one of claims 7 to 10, which is for ELISA measurement.

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