JP4940424B2 - Markers of neuronal oxidative damage and use thereof - Google Patents

Description

  The present invention relates to a marker capable of detecting the degree of oxidative damage in nerve cells and use thereof, and more specifically, to an antibody that specifically recognizes the marker, a measurement kit, a method for measuring oxidative damage, and an inflammatory reaction. The present invention relates to a method for screening related compounds.

  The etiology of neurodegenerative diseases such as Alzheimer's and Parkinson's disease is not clear. On the other hand, various abnormalities of nerve cells related to such neurodegenerative diseases have been reported. For example, it has been reported that the DHA content in the brain of Alzheimer's disease patients is reduced to half that of normal persons (Non-patent Document 1). In addition, dopamine is a catecholamine-based neurotransmitter having an amino group, and it has also been reported that a decrease in dopamine is deeply involved in neurodegenerative diseases such as Parkinson's disease (Non-patent Document 2).

Here, it is known that docosahexaenoic acid (DHA) and arachidonic acid (AA) are abundantly contained in nerve tissues such as the cerebrum and retina, breast milk and the like, and exhibit various physiological activities. Moreover, since these fatty acids are polyunsaturated fatty acids having many double bonds, it is also known that they may be oxidized in vivo to produce lipid peroxides (Non-patent Document 3).
Lipids 26, 421 (1991) J. Med. Chem. 1997,40,2211-2216 “Contemporary Food Chemistry” Sankyo Publishing, Mitsuo Namiki

  However, there has been no investigation or report on the relationship between DHA or AA and dopamine, and further the relationship with neurodegenerative diseases.

  Therefore, the present invention provides a marker suitable for detecting oxidative damage of nerve cells caused by lipid hydroperoxides generated from highly unsaturated fatty acids such as DHA and AA, a method for measuring the damage using the marker, and the marker Another object is to provide a screening method using a specific antibody and the marker. The present invention also provides a marker useful for predicting the onset of a neurodegenerative disease, diagnosing whether or not the neurodegenerative disease is afflicted, the degree of progression of the neurodegenerative disease, and the like, and neurodegeneration using the marker. Another object is to provide diagnosis methods such as disease onset diagnosis, disease diagnosis, and the like, an antibody specific for the marker, and a screening method using the marker.

  The present invention relates to a marker useful for detecting oxidative damage of nerve cells, a test method using the marker, and the like. The present inventors focused on polyunsaturated fatty acids such as DHA and AA and dopamine that are present in large amounts in the brain. And it discovered that the lipid hydropeptide hydroxide produced | generated from these polyvalent saturated fatty acid by the oxidative stress in the living body is related to the fall of the amount of dopamine in the brain. Furthermore, the present inventors can easily reduce the concentration of DHA or AA or dopamine in the brain by measuring the amount of modified dopamine produced by reaction of these lipid hydroperoxides or degradation products thereof with dopamine. It was found that it could be a useful marker for diagnosing the degree of neuronal oxidative damage, neurodegenerative disease, and whether it is in its pre-stage state. That is, according to the present invention, the following means are provided.

According to one form of this invention, the oxidative damage marker compound in a nerve cell represented by the following formula | equation (1) is provided.
(In formula (1), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)

  In this marker compound, R in the formula (1) can be represented by a hexanoyl group or a glutaroyl group, and in the formula (1), R can be represented by a propanoyl group or a succinyl group. . Further, in the formula (1), R is preferably represented by a hexanoyl group or a propanoyl group.

  According to one aspect of the present invention, there is provided a method for examining oxidative damage in nerve cells, the amount of the oxidative damage marker compound according to any one of claims 1 to 4 in a test sample collected from an animal. An inspection method is provided, comprising the step of measuring.

  The test sample in the measurement step is preferably spinal fluid or cerebrospinal fluid. The measurement step is preferably a step using an antibody that specifically recognizes the oxidative damage marker compound. Furthermore, the measurement step is preferably performed by an ELISA method.

  According to one aspect of the present invention, there is provided a method for examining the degree of aging of nerve cells, wherein the amount of the oxidative damage marker compound according to any one of claims 1 to 4 in a test sample collected from an animal is measured. There is provided an inspection method comprising a step of measuring and a step of determining a degree of aging of the nerve cells of the animal based on a measurement result of the measurement step.

  According to one form of this invention, it is a diagnostic method regarding the disease relevant to the oxidative damage of a neuron | cell, Comprising: The oxidative damage in any one of Claims 1-4 in the test sample extract | collected from an animal A step of measuring the amount of the marker compound and a diagnosis of whether the animal is at risk of developing the disease, whether the animal is suffering from the disease, and the degree of progression of the disease based on the measurement result of the measurement step A diagnostic process is provided. In this diagnostic method, the disease can be a neurodegenerative disease.

According to one aspect of the present invention, there is provided an antibody that specifically recognizes any compound selected from the compounds represented by the following formula (1).
(In formula (1), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)

According to one form of this invention, the compound for producing the said antibody selected from the compound represented by the following formula | equation (2) and (3) is provided.
(In formula (2), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)
(In formula (3), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)

According to one form of this invention, the compound for antigens for producing the antibody of Claim 12 selected from the compound represented by the following formula | equation (4) and (5) is provided.
(In the formula (4), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group, and X represents the remainder obtained by removing one amino group from the carrier protein.)
(In Formula (5), R represents a hexanoyl group, a glutaroyl group, a propanoyl group, or a succinyl group, and X represents the remainder obtained by removing one amino group from the carrier protein.)

  According to one aspect of the present invention, there is provided a method for screening a compound that suppresses oxidative damage in nerve cells, comprising the step of administering one or more test compounds to a non-human animal, Measuring the amount of any one of the nerve cell oxidative damage marker compounds in a test sample collected from the administered non-human animal.

  According to one aspect of the present invention, there is provided a method for screening a food that suppresses oxidative damage in nerve cells, the step of administering one or more foods to a non-human animal, and the administration of the food Measuring the amount of any one of the above nerve cell oxidative damage marker compounds in a test sample collected from the non-human animal.

  Hereinafter, a marker compound for oxidative damage of nerve cells, which is one embodiment of the present invention, is a compound represented by the above formula (1). This marker compound is a reaction product of hydroperoxide of AA or DHA, which is one of the main fatty acids in the fatty acid composition of the brain and the like, and dopamine. Therefore, it is an effective marker compound for detecting oxidative damage of nerve cells. Hereinafter, an oxidative damage marker compound according to one aspect of the present invention will be described, and another embodiment of a method for examining oxidative damage of nerve cells, an antibody that specifically recognizes the marker, a measurement kit, and a nerve cell A method for examining the degree of aging, various diagnostic methods for diseases associated with oxidative damage of nerve cells, screening methods and the like will be described.

(Nerve cell oxidative damage marker compound)
The neuronal oxidative damage marker compound of the present invention is represented by the above formula (1). In the above formula (1), both the marker compound I in which R is a hexanoyl group and the marker compound II in which R is a glutaroyl group are considered to be reaction products of AA-derived hydroperoxide and dopamine. The relationship between arachidonic acid and these compounds I and II is shown in FIG. Marker compound I is derived from the non-carboxy terminus of AA, and marker compound II is considered to be derived from the carboxy terminus of AA. These marker compounds I and II are useful for detecting various oxidative damages related to AA in nerve cells, such as AA oxidation, AA content reduction, and AA-induced dopamine content reduction.

  In addition, in the formula (1), both the marker compound III in which R is a propanoyl group and the marker compound IV in which R is a succinyl group are considered to be reaction products of DHA-derived hydroperoxide and dopamine. The relationship between DHA and these compounds III and IV is shown in FIG. Marker compound III is derived from the non-carboxy terminus of DHA, and marker compound IV is believed to be derived from the carboxy terminus of DHA. These marker compounds III and IV are useful for detecting various oxidative damages related to DHA in nerve cells, such as oxidation of DHA, reduction of DHA content, and reduction of dopamine content by DHA.

  Among these, marker compounds I and III are useful as marker compounds because of their high apoptosis-inducing ability.

  For example, marker compound I is prepared by dissolving equimolar amounts of dopamine and hexanoyl anhydride in a mixed solution (1: 1) of phosphate buffer (pH 7.4) and DMF, and reacting at room temperature for about 4 hours. Can be obtained. The marker compound II is prepared by, for example, dissolving equimolar amounts of dopamine and glutaric anhydride in a mixed solution (1: 1) of a phosphate buffer (pH 7.4) and a saturated sodium acetate solution at room temperature. It can be obtained by reacting for about 1 hour. For example, marker compound III is prepared by dissolving equimolar amounts of dopamine and propionic anhydride in a mixed solution (1: 1) of a phosphate buffer (pH 7.4) and a saturated sodium acetate solution. It can be obtained by reacting for about an hour. The marker compound IV is prepared by, for example, dissolving equimolar amounts of dopamine and succinic anhydride in a mixed solution (1: 1) of a phosphate buffer (pH 7.4) and a saturated sodium acetate solution at room temperature. It can be obtained by reacting for about 1 hour.

  The marker compound of the present invention can be used for research use, test use, diagnostic use and screening use of useful drugs and foods related to oxidative damage of nerve cells.

(Testing method for oxidative damage of nerve cells)
The test method of the present invention uses a test sample collected from an animal as a test target. In the present specification, animals include humans and heterogeneous warm-blooded animals other than humans, such as mice, hamsters, rats, guinea pigs, rabbits, dogs, cats, cows, horses, and chickens. Examples of the test sample include liquid samples such as blood, plasma, serum, urine, spinal fluid, and cerebrospinal fluid collected from animals, preferably spinal fluid and cerebrospinal fluid. Moreover, as a test sample, solid samples, such as a cell and a tissue extract | collected from the nervous tissue of an animal, etc. are mentioned. For solid samples such as cells and tissues, the localization of dopamine-modified products in cells or tissues can be detected by immunohistochemical staining. Tissues and such test samples are appropriately pretreated according to the type and necessity.

  This test method includes a step of measuring the oxidative damage marker compound of the present invention in a test sample. The method for measuring the amount of the marker compound in this measurement step is not particularly limited, but considering that the test sample is a biological sample, these marker compounds should be detected (identified) after being separated from contaminants in the test sample. Is preferred. As such an analysis method, various liquid chromatography (LC) such as high performance liquid chromatography (HPLC) can be used. Moreover, LC / MS, LC / MS / MS, etc. which combined liquid chromatography and mass spectrometry can also be used.

  In addition, the amount of the marker compound can be measured using an antibody that specifically recognizes the marker compound, preferably a monoclonal antibody.

(Antibodies that specifically recognize marker compounds)
The antibody of the present invention is an antibody that specifically recognizes any marker compound selected from the marker compounds I to IV represented by the above formula (1). The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody.

(Production of antibody)
A method for producing an antibody against such a marker compound will be described.
(1) Preparation of antigen As an antigen for producing the antibody of the present invention, a conjugate obtained by binding a marker compound and an appropriate carrier such as a protein is preferably used. As the carrier protein, conventionally known carrier proteins are used, among which bovine serum albumin (BSA), ovalbumin, lipoprotein, hemocyanin and the like are preferably used. More preferred is limpet hemocyanin (KLH). Such carrier proteins are readily available commercially. As the carrier, a synthetic polymer such as a polymer or copolymer such as polyamino acid, polystyrene, polyacrylic acid, or polyvinyl can be used.

  A method for obtaining a conjugate of the marker compound and the carrier protein is not particularly limited, but the carboxyl adduct having a carboxyl group introduced into the marker compound and the carrier protein in an appropriate solvent in the presence of an appropriate crosslinking agent. The method of making it react and forming an acid amide bond is mentioned. Examples of the carboxyl adduct include a compound represented by the above formula (2) obtained by introducing cysteine into the 5-position carbon atom of the aromatic rings of the marker compounds I to IV. Moreover, the compound represented by Formula (3) is mentioned. In the formulas (2) and (3), R represents any one of a hexanoyl group, a glutaroyl group, a propanoyl group, and a succinyl group, and corresponds to the marker compounds I, II, III, and IV, respectively.

  For example, as shown in FIG. 3, the carboxyl adduct represented by the above formula (2) contains 100 mM marker compound, 100 mM cysteine and 1 mg / ml in ammonium acetate buffer (0.1 M, pH 5.8). The tyrosinase can be obtained by reacting at room temperature for about 7 hours. An antigenic compound represented by the formula (4) can be obtained by binding the carboxyl adduct to the carboxyl group of cysteine via the acid amide bond with the amino group of the carrier protein via a carrier protein.

  Moreover, the carboxyl adduct represented by the above formula (3) is modified in the same manner as in the case of synthesizing a marker compound using 3,4-dihydroxy-L-phenylalanine as a starting material, as shown in FIG. It can be obtained by synthesizing the body. An antigenic compound represented by the above formula (5) can be obtained by forming an acid amide bond from the carboxyl group derived from phenylalanine of this carboxyl adduct and the amino group of the carrier protein and binding it to the carrier protein. .

  In addition, about the carboxyl adduct about the marker compound having a glutaroyl group and a succinyl group, these carboxyl groups were previously protected by methyl esterification or the like, then reacted with dopamine, and further introduced with protein, It is preferable to hydrolyze with sodium hydroxide or the like to return to the carboxyl group.

  R in Formula (4) and Formula (5) represents any of a hexanoyl group, a glutaroyl group, a propanoyl group, and a succinyl group, and X represents the remainder obtained by removing one amino group from the carrier protein. The amino group forms an acid amide bond with the carboxyl group of the carboxyl adduct. The carrier protein is preferably bovine serum albumin (BSA), ovalbumin, lipoprotein, and hemocyanin, and more preferably limpet hemocyanin (KLH).

  In addition, water-soluble carbodiimide, such as EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), can be used for the coupling | bonding reaction of a carboxyl adduct and carrier protein as a crosslinking agent. Other cross-linking agents may be used. As a solvent for the binding reaction, a buffer solution such as Tris, phosphate, carbonate, acetate or the like can be used. For example, 1 mM to 0.5 M, more preferably 10 mM to 100 mM phosphoric acid. A buffer solution may be mentioned. The pH is not limited as long as the binding reaction proceeds, but is preferably pH 6 to 11, more preferably pH 7 to 9. The reaction time is, for example, 25 to 60 ° C for 1 hour or longer, preferably 30 to 40 ° C for 5 to 40 hours. The obtained conjugate can be finally used as an antigen by employing a general method for separating and purifying proteins such as separation and purification by column and dialysis alone or in combination.

  In order to obtain a conjugate efficiently, the carboxyl group adduct is reacted with a water-soluble carbodiimide such as EDC, and further reacted with sulfo-NHS (N-hydroxysulfosuccinimide) to form an amine-reactive sulfo-NHS ester. In addition, the sulfo-NHS ester can be reacted with a carrier protein. In this case, the reaction solvent for obtaining the sulfo-NHS ester is not particularly limited as long as it is an organic solvent miscible with water, but dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and ethanol are used alone or in combination. Can be used. The reaction conditions are 20 to 60 ° C. for 5 to 40 hours, preferably 20 to 40 ° C. for about 20 to 30 hours. Next, the reaction solution containing the sulfo-NHS ester is then mixed with the carrier protein solution to allow the binding reaction to proceed. As the solution for dissolving the carrier protein, the solvent for the binding reaction already described can be preferably used, and the pH is also the same. The reaction time can be 1 hour or longer, preferably about 2 to 10 hours.

  In addition, the carrier protein and the marker compound may be reacted with a dialdehyde such as glutaraldehyde or a diisocyanate such as hexamethylene diisocyanate.

(2) Production of antibody A warm-blooded animal is immunized with the antigen thus prepared, and finally, a hybridoma that produces the target antibody is screened, and the antibody produced by this hybridoma is purified to obtain the antibody of the present invention. Obtainable. The warm-blooded animal is not particularly limited, and mice, hamsters, rats, guinea pigs, rabbits, dogs, chickens and the like can be used. When the cells to be used as hybridomas are limited, it is preferable to use the same animal as that derived from the myeloma. Since the normally used myeloma is derived from a mouse, it is preferable to use a mouse as a warm-blooded animal.

  In order to immunize a warm-blooded animal with an antigen, a conventionally known method can be employed. For example, the antigen is preferably administered for 7 to 30 days by one or more administration routes selected from subcutaneous injection, intradermal injection, intraperitoneal injection, intravenous injection, intramuscular injection and the like for warm-blooded animals. Is administered about 2 to 10 times at intervals of 12 to 16 days.

  The antigen is used after being dissolved in an appropriate buffer, for example, a phosphate buffer containing 1 type of commonly used adjuvant such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, or physiological saline. Although it is possible, it is not necessary to use such an adjuvant. Here, the adjuvant means a substance that nonspecifically enhances the immune response to the antigen when administered together with the antigen.

  After leaving the warm-blooded animal immunized with the antigen untreated for 7 to 30 days, a small amount of serum of the warm-blooded animal is collected and the antibody titer is measured. Depending on the increase in the antibody titer, additional administration of the antigen can be performed an appropriate number of times. For example, one or two additional administrations are performed at a dose of 0.01 to 1 mg, particularly 0.05 to 0.5 mg. As a method for measuring the antibody titer, a measuring method selected from an enzyme immunoassay, a Western blot method, an agglutination method, a one-way radial immunodiffusion method and the like can be used, but an enzyme immunoassay is preferable.

  1 to 30 days after the last administration to the warm-blooded compound in which the antibody titer was observed, particularly preferably 1 to 7 days later, the spleen or lymph nodes were removed from the immunized warm-blooded animal, and antibody-producing cells were collected from this tissue An antibody-producing hybridoma can be obtained by fusing with a myeloma-derived cell (myeloma) that can be subcultured.

  For the hybridoma, for example, a method described in “Introduction to Monoclonal Antibody Experimental Procedure” (Kodansha Scientific Ando et al. 1991) can be used. That is, antibody-producing cells and myeloma are fused. Sendai virus or polyethylene glycol is used as the induction promoter. The myeloma is preferably the same animal as the animal from which the antibody-producing cells are derived.

  As a cell fusion method actually used, a known technique (J Immunol Method 39: 285-308, 1980) can be used. For example, spleen cells obtained from immunized mice and mouse myeloma cells are fused in the presence of polyethylene glycol, and selected with HAT medium (medium supplemented with hypoxanthine, aminopterin, and thymidine) in which only hybrid cells can grow. After hybrid cells are grown and colonies form, the hybridomas producing the desired antibody can be obtained by screening the antibodies in the culture supernatant. According to the present invention, hybridomas producing such antibodies are also provided.

  Various methods can be employed as a screening method for antibody-producing hybridomas. For the measurement of the antibody titer, for example, a method similar to the measurement of the antibody titer described above can be employed. The enzyme immunoassay is preferable. Specifically, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a marker compound is adsorbed directly or together with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (for cell fusion). When the cells used are mice, an anti-mouse immunoglobulin antibody is used) or a method for detecting a monoclonal antibody bound to a solid phase by adding protein A, a hybridoma on a solid phase adsorbed with an anti-immunoglobulin antibody or protein A Examples include a method of adding a culture supernatant, adding a marker compound labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to a solid phase. Further, by repeating the limiting dilution method, a single hybridoma that produces the antibody of the present invention can be obtained.

  The antibody of the present invention is produced by culturing the thus screened hybridoma in an environment producing a monoclonal antibody (in vitro or in vivo of a warm-blooded animal) and collecting the antibody from a body fluid or a culture solution. Can do. The antibody of the present invention can be performed in the same manner as the separation and purification of a normal polyclonal antibody. That is, immunoglobulin separation and purification methods such as salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, It is carried out according to a specific purification method or the like in which only an antibody is collected with an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the binding is dissociated to obtain the antibody.

  In order to screen for a hybridoma that produces an antibody having a high reaction specificity for the marker compound, it is preferable to examine the specificity for the marker compound and the cross-reactivity for similar compounds by indirect competitive ELISA or direct competitive ELISA.

  The monoclonal antibody of the present invention not only detects human marker compounds I to IV, but also heterologous warm-blooded animals other than humans, such as mice, hamsters, rats, guinea pigs, rabbits, dogs, cats, cows, horses, chickens, etc. The present invention can also be applied to detection of marker compounds I to IV existing in a living body.

  The method for measuring a marker compound using an antibody is not particularly limited as long as it uses a normal antigen-antibody reaction. Radioisotope immunoassay (RIA), enzyme immunoassay (ELISA), immune tissue Examples include chemical methods, fluorescence or luminescence measurement methods, agglutination methods, immunoblot methods, immunochromatography methods (Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol. II IRL Press Oxford (1989)). From the viewpoints of convenience and convenience, ELISA is preferred. Examples of the enzyme used for ELISA include peroxidase, alkaline phosphatase, β-galactosidase, luciferase and the like.

  Examples of the measurement method by ELISA include indirect competitive ELISA and direct competitive ELISA. For example, the indirect competition ELISA can be performed by the following procedure.

(1) The antigen compound, which is an antigen for immobilization, is immobilized on a carrier. As a carrier to be used, a microtiter plate having 96 holes, 48 holes, 192 holes or the like can be used. For immobilization, for example, a buffer containing the immobilization antigen may be placed on a carrier and incubated. The concentration of the antigen in the buffer is usually about 0.01 μg / ml to 100 μg / ml. As the buffer solution, a known solution can be used according to the detection means.

(2) In order to prevent nonspecific adsorption of the protein to the solid phase surface of the carrier, the solid phase surface portion where the antigen for immobilization is not adsorbed is blocked with a protein unrelated to the antigen. As the blocking agent, BSA or skim milk solution, or commercially available Block Ace (Dainippon Pharmaceutical Co., Ltd.) can be used. Blocking is performed by adding the blocking agent to the carrier and, for example, incubating overnight at about 4 ° C. and then washing with a washing solution. Although there is no restriction | limiting in particular as a washing | cleaning liquid, The same buffer solution as said (1) can be used.

(3) The test sample containing various concentrations of the antigenic compound and the antibody solution of the present invention are added to the solid phase surface treated in the above (1) and (2), and the antibody is added to the immobilized antigen and the test sample. The marker compound is allowed to react competitively to produce an immobilized antigen-antibody complex and a marker compound-antibody complex. The reaction can be carried out at 10 ° C. to 40 ° C., preferably 25 ° C. to 37 ° C. for about 0.5 to several hours.

(4) By measuring the amount of the immobilized antigen-antibody complex, the amount of the marker compound in the sample can be determined from a calibration curve prepared in advance. The amount of the immobilized antigen-antibody complex can be measured by adding an enzyme-labeled secondary antibody (an antibody recognizing the antibody of the present invention). For example, when a mouse monoclonal antibody is used as the antibody of the present invention, it is desirable to react with an antibody of the present invention bound to a carrier using an anti-mouse-goat antibody labeled with an enzyme (for example, peroxidase or alkaline phosphatase). The reaction may be performed under the same conditions as in (3) above. After the reaction, wash with buffer.

(5) The amount of the marker compound can be calculated from the calibration curve by adding a chromogenic substrate solution that reacts with the labeling enzyme of the secondary antibody bound to the carrier and measuring the absorbance. When using peroxidase as an enzyme that binds to the secondary antibody, for example, use a chromogenic substrate solution containing hydrogen peroxide and 3,3 ′, 5,5′-tetramethylbenzidine or o-phenylenediamine. Can do. Usually, after adding a chromogenic substrate solution and reacting at room temperature for about 10 minutes, the enzyme reaction is stopped by adding sulfuric acid. When 3,3 ', 5,5'-tetramethylbenzidine is used, the absorbance at 450 nm is measured. When o-phenylenediamine is used, the absorbance at 492 nm is measured. In order to correct the background value, it is desirable to simultaneously measure the absorbance at 630 nm. When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, for example, color development is performed using p-nitrophenyl phosphate as a substrate, the NaOH reaction is added to stop the enzyme reaction, and the absorbance at 415 nm is measured. It is done.

  For the absorbance of the reaction solution to which no marker compound is added, the rate of decrease in the absorbance of the solution reacted with the antibody by adding the marker compound is calculated as the inhibition rate. The concentration of the marker compound in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which a known concentration of alachlor is added.

  In addition, the marker compound can be measured directly by competitive ELISA as follows. That is, instead of the solid phased antigen, the antibody of the present invention is solid phased on a carrier, and instead of the antibody mixed with the test sample, an enzyme-linked hapten in which a marker compound and an enzyme are bound is used, and no secondary antibody is used. The amount of the immobilized antibody-enzyme linked hapten complex was developed in the same manner as in the indirect competitive ELISA, and the absorbance was measured in advance. The amount of the marker compound in the test sample can be determined from the prepared calibration curve.

  In addition, in the measuring method of this invention, after pre-processing according to a test sample and using it as a sample, it is used for indirect competitive ELISA, direct competitive ELISA, and other measuring methods.

  As described above, by measuring the amount of such a marker compound in the test sample, various oxidative damage states in nerve cells such as the degree of oxidation and content of DHA and / or AA, and the content of dopamine are decreased. Can be detected. Therefore, by using the test method of the present invention, a test method for the degree of aging of nerve cells, a diagnostic use, and a screening method for useful drugs and foods are provided.

(Examination method of neuronal aging degree, etc.)
For example, there is provided an inspection method comprising a main measurement step in the present inspection method, and a determination step of determining the degree of aging of the nerve cells of the animal based on the measurement result of the main measurement step. According to the present inventors, the degree of oxidative damage (amount of marker compound) in various aspects of such nerve cells is considered to be related to the degree of aging of nerve cells. That is, if the amount of the marker compound is large, it can be said that aging is more advanced. In determining the degree of aging, for example, the relationship between the age or age of a test animal such as a human and the marker amount is obtained in advance, and the marker compound amount detected from the test sample and the marker compound amount corresponding to the age of the test animal It is possible to determine whether aging is more advanced or whether aging is slower. In addition, for example, by periodically measuring the amount of the marker compound for the test animal, it can be determined whether or not aging has progressed in the nerve cells. Furthermore, by determining the degree of aging of the nerve cells of the test animal individual, anti-aging measures and treatments for improving or preventing a physiological state in which oxidative damage occurs can be performed.

  And a diagnosis step comprising diagnosing one of the present measurement step and the risk of developing a disease associated with oxidative damage of nerve cells in an animal, whether the disease is affected by the disease, and the degree of progression of the disease. A method is provided. According to the present inventors, the degree of oxidative damage (amount of marker compound) in various aspects of such nerve cells depends on the onset and progression of diseases associated with these damages, in other words, diseases associated with marker compounds. It seems to be related to. That is, if the amount of the marker compound is large, it can be said that the risk of onset is high, the disease is already affected, or the disease is considerably advanced. Examples of such diseases include neurodegenerative diseases such as Alzheimer's disease, Pick's disease, Parkinson's syndrome (Parkinson's disease, symptomatic parkinsonism), progressive supranuclear palsy, Huntington's chorea, Sydenham chorea, multisystem atrophy. , Hereditary spinocerebellar degenerative disease, cortical cerebellar atrophy, spinal cerebellar degeneration such as late cortical cerebellar I reduction, amyotrophic lateral sclerosis, progressive bulbar paralysis, spinal muscular atrophy, sub Acute associated spinal degeneration, bulbar spinal muscular atrophy and the like. Among them, Alzheimer's disease and Parkinson's syndrome are mentioned.

  In the diagnosis of disease onset risk, disease diagnosis, and degree of progression of disease state, for example, in humans, the relationship between the presence or absence of a disease (normal person and diseased person) and the amount of marker compound, age (age) and amount of marker compound , The relationship between sex difference and the amount of marker compound, the relationship between the degree of disease progression (mild, moderate, severe) and the amount of marker compound, etc., and the amount of marker compound detected from the test sample By selecting and comparing the amount of marker compound to be compared in consideration of the age, presence / absence of symptom, sex difference, degree of progression, etc. of the test animal, the onset risk, the presence / absence of the disease, and the degree of progression of the disease state can be diagnosed. In addition, for example, by periodically measuring the amount of the marker compound for a subject, it is possible to diagnose whether there has been a change in the onset risk, onset, progression of the disease state, or the like.

(Screening method)
The screening method for a compound that suppresses or promotes oxidative damage in nerve cells of the present invention comprises a step of administering one or more test compounds to a non-human animal and the non-human animal administered with the test compound. Measuring the amount of any compound selected from marker compounds I to IV in a test sample to be collected. According to this screening method, when a test compound is administered to a non-human animal, an increase or decrease in the amount of marker compounds I to IV can be detected. Therefore, it can be evaluated whether the test compound promotes or suppresses oxidative damage of nerve cells. As a result, according to the screening method of the present invention, a compound that regulates (suppresses or promotes) oxidative damage of nerve cells can be screened.

  In particular, the compound that suppresses oxidative damage of nerve cells obtained by this screening method is used as a drug for the prevention or treatment of diseases associated with marker compounds I to IV or diseases associated with oxidative damage of nerve cells. be able to.

  The present invention also relates to a method for screening a food for suppressing oxidative damage in nerve cells, the step of administering one or more kinds of food to a non-human animal, and the non-human animal administered with the test compound. Measuring the amount of any compound selected from marker compounds I to IV in a test sample collected from the test sample. According to this screening method, when food is administered to a non-human animal, whether the administered food promotes oxidative damage of nerve cells by detecting an increase or decrease in the amount of marker compounds I to IV. Can be evaluated. As a result, foods that can suppress oxidative damage to nerve cells can be screened. Such foods are provided as foods that prevent and ameliorate physiological conditions and diseases caused by oxidative damage of nerve cells.

  In this screening method, it is preferable to use, as a non-human animal, a non-human animal in which oxidative damage of nerve cells is promoted for screening of a compound or food that suppresses oxidative damage of nerve cells. Such non-human animals can be obtained by genetic engineering modification, artificial mating, or searching from mutants. Typically, there are Alzheimer model mice, Parkinson model mice, and the like.

(Measurement kit)
The measurement kit of the present invention contains an antibody that specifically recognizes the marker compound of the present invention. For this reason, the marker compound in the test sample can be easily measured. The measurement kit further contains a labeled secondary antibody or a labeled marker compound hapten (antigen), a buffer solution, a detection reagent and / or a marker compound standard solution, depending on the measurement method. A preferred kit is one that can be used in the ELISA method, and can contain a carrier that holds an immobilized antigen, the antibody of the present invention, an enzyme-labeled secondary antibody, a detection reagent, and the like.

  Since the marker compound specifically recognized by the antibody of the present invention is produced by the reaction of hydroperoxide, which is a peroxide of AA or DHA, and dopamine, according to this measurement kit, By measuring the marker compound, it is possible to detect the oxidative damage and the degree of nerve cells in the collection site of the test sample or the collected individual.

(Immobilized body)
The immobilized body of the present invention comprises a solid phase carrier and the antibody of the present invention immobilized on the solid phase carrier. The solid phase carrier is not particularly limited in form such as a substrate shape or a bead shape, and the material thereof may be a conventionally known material such as glass, ceramics, plastic, or metal. Furthermore, it may be porous or dense. The form of immobilization of the antibody of the present invention on the solid phase carrier is not particularly limited as long as it is immobilized by a conventionally known technique that can hold a protein such as an antibody on the solid phase, such as covalent bond or electrostatic bond. In the immobilized body of the present invention, the antibody of the present invention is preferably immobilized on a substrate-like solid phase carrier together with other antibodies in advance with positional information. Such an immobilized body of the present invention can take the form of an antibody chip in which a plurality of antibodies are immobilized on a single solid substrate carrier.

EXAMPLES Specific examples of the present invention will be described below as examples, but the present invention is not limited to these examples.
Example 1
In this example, hexanoyl dopamine (HED: the following formula (6)), glutaroyl dopamine (GLD: the following formula (7)), propanoyl dopamine (PRD: the following formula (8)), which are the marker compounds of the present invention. And succinyl dopamine (SUD: the following formula (9)) were synthesized respectively.

(1) HED
After adding 63.9 mg (0.337 mmol) of dopamine and 72.3 mg (0.337 mmol) of hexanoyl anhydride to 4.4 ml of a mixed solution (1: 1) of DMF / phosphate buffer (pH 7.4), a stirrer was added. And allowed to react at room temperature for 4 hours. This reaction solution was separated using LC / MS / MS, and the molecular weight was confirmed (m / z 252.2 → 137.2). Moreover, the structure was determined using ¹ H-NMR and ¹³ C-NMR. The results are shown in Table 1.

(2) GLD
After adding 95 mg (0.5 mmol) of dopamine to 5 ml of a phosphate buffer (pH 7.4) / saturated sodium acetate aqueous solution (1: 1), 57 mg (0.5 mmol) of glutaric anhydride dissolved in a small amount of pyridine Was added at 1 drop / second. And it was made to react at room temperature for 1 hour using the stirrer. Methanol was added to this reaction solution, pyridine was skipped using an evaporator, and fractionated using HPLC. Further, the molecular weight of the fraction was confirmed using LC / MS / MS (m / z 268.2 → 137.2). Moreover, the structure was determined using ¹ H-NMR and ¹³ C-NMR. The results are shown in Table 2.

(3) PRD
After adding 95 mg (0.5 mmol) of dopamine to 5 ml of a phosphate buffer (pH 7.4) / saturated aqueous sodium acetate solution (1: 1), 65 mg (0.5 mmol) of propionic anhydride dissolved in a small amount of pyridine Was added at 1 drop / second. And it was made to react at room temperature for 1 hour using the stirrer. Methanol was added to this reaction solution, pyridine was skipped using an evaporator, and fractionated using HPLC. Further, the molecular weight of the fraction was confirmed using LC / MS / MS (m / z 210.1 → 137.2). Moreover, the structure was determined using ¹ H-NMR and ¹³ C-NMR. The results are shown in Table 3.

(4) SUD
After adding 95 mg (0.5 mmol) of dopamine to 5 ml of a phosphate buffer (pH 7.4) / saturated aqueous sodium acetate solution (1: 1), 49 mg (0.5 mmol) of succinic anhydride dissolved in a small amount of pyridine Was added at 1 drop / second. And it was made to react at room temperature for 1 hour using the stirrer. Methanol was added to this reaction solution, pyridine was skipped using an evaporator, and fractionated using HPLC. Further, the molecular weight of the fraction was confirmed using LC / MS / MS (m / z 254.1 → 137.2). Moreover, the structure was determined using ¹ H-NMR and ¹³ C-NMR. The results are shown in Table 4.

In identifying these marker compounds, the conditions of LC / MS / MS (Applied Biosystem API 2000 HPLC-MS / MS system) were as follows.
[LC-MS / MS conditions for HED and GLD]
Column: Develosil ODS-HG-3 (2 φ × 50 mm)
Mobile phase: A; 100% H ₂ O / 0.1% HCOOH
B: 100% CH3CN / 0.1% HCOOH
Flow rate: 0.2 ml / min
Ion source: Turbo Spray Ionization positive
Gradient program

[LC-MS / MS conditions for PRD and SUD]
The LC-MS / MS conditions for identification of HED etc. were the same except for the settings for gradient program and fragment detection.
Gradient program

(Example 2)
(In vitro marker compound formation reaction)
In this example, it was confirmed in vitro whether various marker compounds were produced from lipid hydroperoxide and dopamine. The LC / MS / MS conditions were the same as in Example 1.
(1) HED
Since HED is produced from a ω-6 polyunsaturated fatty acid containing arachidonic acid, 13-hydroxyperoxydecadadenoicacid (13-HPODE), which is a lipid hydroperoxide derived from ω-6 linoleic acid, was used. The reaction was carried out at 37 ° C. in a phosphate buffer (pH 7.4) so that the final concentration was 10 mM 13-HPODE and 2 mM dopamine, and the reaction was stopped at −80 ° C. When the reaction solution was subjected to LC / MS / MS, HED could be detected.

(2) GLD
After mixing 100 μl of 100 mM arachidonic acid, 0.8 mg of lipoxygenase and 11 mg of borate buffer (pH 9.0), the mixture was stirred for 30 minutes while blowing oxygen. The pH was adjusted to 4.0 or lower by adding 1N HCl, followed by extraction with 20 ml of a chloroform / methanol mixture (1: 1), and the chloroform layer was collected, dehydrated and dried. This was redissolved with 100 μl ethanol to obtain a 100 mM arachidonic acid hydroperoxide fraction.

  The reaction was carried out at 37 ° C. in a phosphate buffer (pH 7.4) so that the final concentration was 10 mM arachidonic acid hydroperoxide fraction and 2 mM dopamine, and the reaction was stopped at −80 ° C. When LC / MS / MS was performed on the reaction solution, GLD was detected.

(3) PRD
The reaction was carried out at 37 ° C. in a phosphate buffer (pH 7.4) so that the final concentrations were 10 mM DHA and 2 mM dopamine, and the reaction was stopped at −80 ° C. When the reaction solution was subjected to LC / MS / MS, PRD could be detected.

(4) SUD
The reaction was carried out at 37 ° C. in a phosphate buffer (pH 7.4) so that the final concentrations were 10 mM DHA and 2 mM dopamine, and the reaction was stopped at −80 ° C. When the reaction solution was subjected to LC / MS / MS, SUD could be detected.

(Example 3)
In this example, the presence of various marker compounds in the rat brain was confirmed.
(Sample preparation)
Six week old female rat females were used. The brain was removed and half of the whole brain was used for homogenate. The brain for homogenate was added to an extract (methanol: 5 mM BHT: 250 mM EDTA mixture (90: 5: 5 by volume)) 4 times the wet weight, and then homogenated to recover the methanol layer. Methanol was distilled off to dryness, and the dried product was redissolved with methanol, and the fraction near the detection time of the marker compound was collected by HPLC, and then the collected fraction was dried and dried again. 5 was analyzed for the presence or absence of a marker compound using LC / MS / MS, and the results are shown in FIG.

  The HPLC conditions for fractionation were as follows, and LC-MS / MS was carried out using the same conditions as in Example 1 and using an internal standard. Moreover, the structure etc. of the internal standard substance used for the following identification are shown in FIG.6 and FIG.7.

[Preparative HPLC conditions]
Column: Develosil ODS-HG-5 (4.6 φ × 250 mm)
Mobile phase: A; 100% H ₂ O / 0.01% CH ₃ COOH
B: 100% CH ₃ CN / 0.01% CH ₃ COOH
Flow rate: 0.8 ml / min
Detector (UV): 280 nm
Grangeent program

  As shown in FIG. 5, all marker compounds of HED, GLD, PRD and SUD were detected from the rat brain. From the above, it was confirmed that these marker compounds were also produced in vivo.

Example 4
In this example, the apoptosis-inducing activity of various marker compounds was evaluated.
For the evaluation, a cultured cell line derived from human neuroblastoma (SH-SY5Y cells) was used. Cells were seeded in a 6-well flask at approximately 5 × 10 ⁵ cells / well, cultured in an incubator (37 ° C., 5% CO ₂ ) overnight, and then replaced with a medium containing each marker compound. . Each medium was prepared so that the marker compound concentration was 100 μM at the final concentration. Cytotoxicity was evaluated by performing nuclear staining 24 hours later. Propidium Iodide (PI), Hoechst 33258, and pentahydrate (bis-benzimide) were used for nuclear staining. PI evaluated dead cells and Hoechst evaluated both dead and live cells. Therefore, cytotoxicity was evaluated by determining PI / Hoechst. The evaluation method was performed by observing with a microscope, and the evaluation was performed by counting the number of cells stained with each reagent. In addition, it evaluated similarly in the medium which added DMSO and DA instead of the marker compound, and the additive-free medium. The results are shown in FIG.

  As shown in FIG. 8, PI / Hoechst was 60% or more in HED, and about 30% in PRD.

It is a figure which shows the relationship between the marker compounds I and II by reaction of arachidonic acid hydroperoxide and dopamine. It is a figure which shows the relationship with marker compound III and IV by reaction of docosahexaenoic acid hydroperoxide and dopamine. It is a figure which shows the scheme which obtains the compound for antigens represented by Formula (4) from the carboxyl adduct represented by Formula (2). It is a figure which shows the scheme which obtains the compound for antigens represented by Formula (5) from the carboxyl adduct represented by Formula (3). It is a figure which shows the LC / MS / MS chart which detected the marker compound in a synthetic rat brain. It is a figure which shows the internal standard substance with respect to HED and GLD. It is a figure which shows the internal standard substance with respect to PRD and SUD. It is the graph which evaluated the apoptosis induction activity of the marker compound.

Claims (16)

An oxidative damage marker in nerve cells represented by the following formula (1).
(In formula (1), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)   The oxidative damage marker according to claim 1, wherein R in the formula (1) is represented by a hexanoyl group or a glutaroyl group.   The oxidative damage marker according to claim 1, wherein R in the formula (1) is represented by a propanoyl group or a succinyl group.   The oxidative damage marker according to claim 1, wherein R in the formula (1) is represented by a hexanoyl group or a propanoyl group. A method for examining oxidative damage in nerve cells,
The test | inspection method provided with the process of measuring the quantity of the oxidative damage marker in any one of Claims 1-4 in the test sample extract | collected from an animal.   The test method according to claim 5, wherein the test sample in the measurement step is spinal fluid or cerebrospinal fluid.   The test method according to claim 5 or 6, wherein the measurement step is a step of using an antibody that specifically recognizes the oxidative damage marker.   The inspection method according to claim 7, wherein the measurement step is performed by an ELISA method. A test method for the risk of developing a disease associated with oxidative damage of nerve cells,
Measuring the amount of the oxidative damage marker according to any one of claims 1 to 4 in a test sample collected from an animal;
A determination step of the onset risk of the disease in the animal to determine the constant based on the measurement result of the measuring step,
An inspection method comprising: The test method according to claim 9 , wherein the disease is a neurodegenerative disease. An antibody that specifically recognizes any compound selected from compounds represented by the following formula (1).
(In formula (1), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.) The antigen preparation agent for preparing the antigen used for producing the antibody of Claim 11 selected from the compound represented by the following formula | equation (2) and (3).
(In formula (2), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.)
(In formula (3), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group.) The antibody production agent for producing the antibody of Claim 11 selected from the compound represented by the following formula | equation (4) and (5).
(In formula (4), R represents a hexanoyl group, a glutaroyl group, a propanoyl group or a succinyl group, and X represents the remainder obtained by removing one amino group from the carrier protein.)
(In Formula (5), R represents a hexanoyl group, a glutaroyl group, a propanoyl group, or a succinyl group, and X represents the remainder obtained by removing one amino group from the carrier protein.) A method for screening a compound that suppresses oxidative damage in a nerve cell, comprising:
Administering one or more test compounds to a non-human animal;
Measuring the amount of the neuronal oxidative damage marker compound according to any one of claims 1 to 4 in a test sample collected from the non-human animal administered with the test compound;
A screening method comprising: A method for screening foods that suppresses oxidative damage in nerve cells,
Administering one or more foods to a non-human animal;
Measuring the amount of the nerve cell oxidative damage marker compound according to any one of claims 1 to 4 in a test sample collected from the non-human animal administered with the food;
A screening method comprising: A compound represented by the following formula (1).
(In formula (1), R represents a hexanoyl group or a glutaroyl group.)