JPWO2009069528A1 - Evaluation method of fatigue level and kit for evaluating fatigue level - Google Patents

Abstract

It is an object of the present invention to provide a fatigue evaluation method based on a new fatigue biomarker and a kit for evaluating fatigue. A fatigue evaluation method based on the knowledge that extracellular secretion of irreversible oxidized peroxiredoxin reflects the oxidative stress state of a cell and a kit for evaluating fatigue are provided.

Description

  The present invention relates to a fatigue evaluation method and a kit for evaluating fatigue.

  Fatigue is one of the three major biological alarms along with pain and heat, and is a biological signal that informs you of physical abnormalities. Fatigue is caused by physical or mental stress and is often accompanied by a feeling of fatigue such as loss of concentration, loss of motivation, increased discomfort, and induction of sleepiness. Furthermore, fatigue accumulation and chronic fatigue are thought to be involved in the development of various diseases including lifestyle-related diseases such as arteriosclerosis, myocardial infarction, diabetes, and cancer.

  According to the announcement by the Ministry of Health, Labor and Welfare, the number of work-related injuries for brain and heart disease due to overwork is over 310 per year, and about 9 thousand of the more than 34,000 suicides per year are workers (Heisei 15 As of fiscal year). In addition, overtime work hours tend to increase, and over 60% of workers feel strong anxiety and stress at work. Against this background, emphasis is placed on strengthening measures against overwork and mental health at the policy level, and at the individual level, including workers, it is important to understand and be aware of the level of fatigue. There is a growing need for self-care.

  Conventional methods for evaluating stress and stress on the body during work include psychological evaluation methods based on subjective symptoms, task evaluation methods such as flicker, electrophysiological measurement methods that measure brain waves and heartbeats, and stress responses contained in body fluids There are biochemical measurement methods that measure substances and immune response substances. Among these, biochemical measurement methods can be given as an objective and quantitative evaluation method. So far, for example, from a concentration of chromogranin in a body fluid, a method for evaluating a physiological state correlated with the secreted amount of chromogranin (Patent Document 1), a method for evaluating fatigue using an acylcarnitine concentration in a body fluid as an index ( A biochemical measurement method such as Patent Document 2) is disclosed.

Japanese Patent Laid-Open No. 11-23572 JP-A-2005-70024 J. et al. Biol. Chem. , 277, 19396-19401 (2002)

  However, in conventional biochemical measurement methods, substances that respond to various stresses that cause fatigue are mainly used as indicators. That is, these indicators were mainly mediators of stress signals or output substances. Therefore, the present inventor considered that if the state of stress itself that causes fatigue can be measured, a new biomarker for fatigue can be provided.

  The mechanism that causes fatigue has not yet been elucidated, but a mechanism involving oxidative stress has been proposed as a model. It is known that reactive oxygen species cause oxidative stress such as abnormal molecular function and cell / tissue damage by reacting nonspecifically with biomolecules such as proteins, lipids, and nucleic acids. If the balance of the mechanism for removing oxidative stress that maintains the redox state at the cell / tissue level is lost, an oxidative stress state in which chemically unstable reactive oxygen species cannot be removed is obtained. It is considered that the function of such an in-vivo defense system is reduced under physical or mental stress.

  Based on these findings, the present inventor has become a clue to elucidating the mechanism of fatigue by detecting the state of oxidative stress, and more objective and comprehensive in combination with conventional biochemical measurement methods. We thought that it was possible to evaluate the degree of fatigue. However, conventionally, it has been difficult to quantitatively evaluate the reactive oxygen species and oxidative stress removal mechanism generated in the living body because of its high reactivity and reversible reaction system. The present invention has been made in view of the above-described problems of the prior art, identifies an objective index of oxidative stress, provides a new fatigue biomarker, and fatigue degree based on the new fatigue biomarker It aims at providing the kit for evaluating the evaluation method and fatigue degree.

  As a result of investigating the influence of oxidative stress caused by hydrogen peroxide on nerve cells, the present inventors have found that the secretion amount of irreversible oxidized peroxiredoxin increases depending on the oxidative stress load. It was. These findings indicate that extracellular secretion of irreversible oxidized peroxiredoxin reflects the oxidative stress state of the cell, and thus suggests that it may be effective as an index that reflects the degree of fatigue. Yes. Based on this finding, the present inventor has made it possible to provide a fatigue evaluation method using peroxiredoxin as an index and a kit for evaluating fatigue, and completed the present invention.

  That is, the present invention includes a step of measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from a subject, a step of determining fatigue based on the abundance of irreversible oxidized peroxiredoxin, A method for evaluating the degree of fatigue is provided. The evaluation method of the present invention is based on the knowledge that the extracellular secretion of irreversible oxidized peroxiredoxin reflects the oxidative stress state of cells. Such a molecular mechanism has been newly discovered by the present inventor, and this makes it possible to evaluate the degree of fatigue in a minimally invasive, simple and quantitative manner.

  The evaluation method of the present invention also comprises a step of measuring the ratio of the amount of irreversible oxidized peroxiredoxin and the amount of peroxiredoxin in a body fluid collected from a subject, And a step of determining the degree of fatigue based on a ratio between the abundance and the abundance of peroxiredoxin. Thus, by considering the ratio between the amount of irreversible oxidized peroxiredoxin and the amount of peroxiredoxin, more accurate, taking into account the balance of the redox state of peroxiredoxin Can be highly evaluated.

  In the evaluation method of the present invention, the step of measuring the abundance of the irreversible oxidized peroxiredoxin includes the following steps: immobilizing the irreversible oxidized peroxiredoxin in a body fluid collected from a subject; A step of forming an immune complex between a solid-phased irreversible oxidized peroxiredoxin and a labeled first antibody, wherein the first antibody is specific to the irreversible oxidized peroxiredoxin Recognizing and labeling the antibody, and detecting the signal intensity derived from the label of the first antibody in the immune complex and measuring the abundance of irreversible oxidized peroxiredoxin; Are preferably provided.

  In the evaluation method of the present invention, the step of measuring the abundance of the irreversible oxidized peroxiredoxin includes the following steps: a step of immobilizing the irreversible oxidized peroxiredoxin in a body fluid collected from a subject And an immobilized complex of irreversible oxidized peroxiredoxin and a first antibody, wherein the first antibody is specific to irreversible oxidized peroxiredoxin. A step of forming an immune complex of the above-mentioned immune complex and a second molecule, wherein the second molecule specifically binds the first antibody. A step of recognizing and labeling, and a step of detecting the signal intensity derived from the labeling of the second molecule in the above-mentioned three immune complexes and measuring the abundance of irreversible oxidized peroxiredoxin And preferably Arbitrariness.

  By providing the process of the present invention, the abundance of the irreversible oxidized peroxiredoxin can be easily and quantitatively measured, and the fatigue level can be evaluated.

  Further, in the evaluation method of the present invention, the step of measuring the abundance of the irreversible oxidized peroxiredoxin was collected from the following steps: a step of immobilizing a standard irreversible oxidized peroxiredoxin, and a solid phase In the presence of irreversible oxidized peroxiredoxin in a body fluid, the step of forming an immunocomplex of the solid-phased standard irreversible oxidized peroxiredoxin and the labeled first antibody, The first antibody specifically recognizes irreversible oxidized peroxiredoxin and detects the signal intensity derived from the labeled antibody and the labeling of the first antibody in the immune complex; And measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from a subject.

  Further, in the evaluation method of the present invention, the step of measuring the abundance of the irreversible oxidized peroxiredoxin was collected from the following steps: a step of immobilizing a standard irreversible oxidized peroxiredoxin, and a solid phase In the presence of irreversible oxidized peroxiredoxin in a body fluid, the step of forming an immune complex between the solid-phased standard irreversible oxidized peroxiredoxin and the labeled first antibody, The first antibody specifically recognizes irreversible oxidized peroxiredoxin and is a labeled antibody, and a step of forming a ternary immune complex of the immune complex and a second molecule. Wherein the second molecule is derived from the step of specifically recognizing and labeling the first antibody and the label of the second molecule in the tripartite immune complex. Detect signal intensity A step of measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from a subject is preferably provided with a.

  By providing the process of the present invention, the amount of irreversible oxidized peroxiredoxin can be easily and quantitatively measured even when a sample containing a large amount of contaminating proteins is used, and the degree of fatigue is evaluated. It becomes possible.

  The present invention provides a kit for evaluating the degree of fatigue, comprising a solid phase for performing the above-described evaluation method, a first antibody, and instructions for use. The present invention also provides a kit for evaluating the degree of fatigue, comprising a solid phase, a first antibody, a second molecule, and instructions for use for performing the above evaluation method. The kit of the present invention is based on the knowledge newly discovered by the present inventor that the secreted amount of irreversible oxidized peroxiredoxin reflects the oxidative stress state of cells. This makes it possible to evaluate the degree of fatigue simply and quantitatively based on the amount of irreversible oxidized peroxiredoxin. When measuring the ratio of the amount of irreversible oxidized peroxiredoxin to the amount of peroxiredoxin, the amount of irreversible oxidized peroxiredoxin is measured using this kit. The abundance is measured using known methods.

  According to the fatigue level evaluation method of the present invention, the fatigue level is minimally invasive, simple and quantitative based on the knowledge that the extracellular secretion of irreversible oxidized peroxiredoxin reflects the oxidative stress state of cells. Can be evaluated. Moreover, according to the kit for evaluating the fatigue level of the present invention, the fatigue level can be easily and quantitatively evaluated. Quantitative measurement of the oxidative stress state in the oxidative stress removal mechanism leads to the provision of a new biomarker of fatigue, which makes it possible to objectively assess the degree of fatigue, especially for chronic fatigue and unconscious fatigue. An evaluation can be given and it is expected to contribute to preventive medicine. It is also expected as an index in anti-fatigue food and drug development.

It is a graph which shows the influence of hydrogen peroxide which gives to the cell activity of cultured mouse neuroblastoma (Neuro-2a). Pre-cultured Neuro-2a was treated with hydrogen peroxide at various concentrations for 1 hour, and MTT assay was performed after washing. It is a graph which shows the irreversible oxidation type peroxiredoxin secretion amount in the oxidative stress load of a cultured mouse neuroblastoma (Neuro-2a). 0.05 mM hydrogen peroxide treatment was carried out for 60 minutes, and the amount of irreversible oxidized peroxiredoxin in the culture supernatant was quantified by an antigen-immobilized ELISA method. It is a figure corresponded to the gel-stained image of two-dimensional electrophoresis which shows the influence of hydrogen peroxide with respect to the secreted protein of a cultured mouse neuroblastoma (Neuro-2a). The upper image is a migration image of a control without addition of hydrogen peroxide, and the lower image is an electrophoresis image under conditions where hydrogen peroxide is added. It is a graph which shows the weight change by various processes. It is a graph which shows the irreversible oxidation type peroxiredoxin density | concentration in rat plasma.

  Hereinafter, a fatigue evaluation method and a kit for evaluating fatigue according to preferred embodiments of the present invention will be described in detail. Note that the present invention is not limited to this.

  First, irreversible oxidized peroxiredoxin and peroxiredoxin, which serve as an index for evaluating the degree of fatigue, will be described.

(Peroxiredoxin)
A plurality of oxidoreductases and electron donors are involved in the mechanism for removing oxidative stress. Conventionally, glutathione peroxidase and catalase were known as oxidoreductases, but peroxiredoxin was identified as a new member. In peroxiredoxin, cysteine present in the active center reversibly transfers between a reduced form (—SH) and an oxidized form (disulfide bond formation between dimers), and catalyzes a reaction for removing active oxygen. There are subtypes 1 to 6 in peroxiredoxins, and the active center cysteine which is an oxidation site and its neighboring amino acid sequences are conserved in many subtypes. Among these, mouse and human peroxiredoxin 2 (SEQ ID NOs: 1 and 2, GenBank accession numbers: AAC35744 and CAA80269) are molecules having a molecular weight of approximately 22 kDa consisting of 198 amino acid residues, and the cysteine present in the active center is It corresponds to the cysteine at the 51st residue (hereinafter referred to as 51Cys). It is known that peroxiredoxin 2 is mainly localized in the cytoplasm in cells.

(Irreversible oxidized peroxiredoxin)
Under excessive oxidative stress, the cysteine present at the active center of peroxiredoxin is excessively oxidized, via sulfenic acid (—SOH) and sulfinic acid (—SO ₂ H), to sulfonic acid (—SO ₃ H). Change. Of these, sulfenic acid is reversible, but sulfinic acid and sulfonic acid cannot be returned to the reduced form with a reducing agent such as DTT. Therefore, sulfinated peroxyredoxin and sulfonated peroxyredoxin are irreversible oxidized forms. It can be called peroxiredoxin (Non-patent Document 1).

(Fatigue evaluation method)
Next, the fatigue level evaluation method according to this embodiment will be described.
The fatigue level evaluation method according to the present embodiment includes a step of measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from a subject, and a fatigue level based on the abundance of irreversible oxidized peroxiredoxin. Determining.

  In another embodiment, the method for assessing fatigue level comprises the steps of measuring the ratio of the amount of irreversible oxidized peroxiredoxin and the amount of peroxiredoxin in a body fluid collected from a subject, and the irreversible oxidized type Determining the degree of fatigue based on the ratio between the amount of peroxiredoxin and the amount of peroxiredoxin.

  The subject is preferably a human. In addition, the subject may be a mammal, and in this case, it is preferably a domestic animal such as a cow, pig, horse or sheep, or a mouse, rat or rabbit. The body fluid collected from the subject is preferably blood, urine, saliva or the like. This makes it possible to evaluate the degree of fatigue in a minimally invasive, simple and quantitative manner. The measurement value obtained in the measuring step may be a relative value in the measurement system, or an absolute value calculated using the standard as a control. Specific measurement methods in the present embodiment include, but are not limited to, an antigen-immobilized immunoassay method using an anti-irreversible oxidized peroxiredoxin antibody, an indirect competition method, which will be described later, A measurement method by a direct competition method or a sandwich method, a measurement method combining an immunoprecipitation method and an immunoassay method, a measurement method by capillary isoelectric focusing using a labeled antibody, or the like may be used. Then, in the step of determining the degree of fatigue, it is preferable to statistically process values obtained from the healthy group and the fatigue group, and to determine based on which one corresponds. Further, the measurement may be performed periodically and compared with the normal value of the target.

(Antigen-immobilized immunoassay, indirect competitive immunoassay, and kit)
The antigen-immobilized immunoassay method according to the embodiment of the present invention is appropriately performed using an antibody that specifically recognizes irreversible oxidized peroxiredoxin, an enzyme immunoassay (ELISA method), a fluorescent immunoassay ( Known immunological measurement methods such as FIA method and radioimmunoassay method (RIA method) can be applied.

Here, the anti-irreversible oxidized peroxiredoxin antibody is an antibody that can specifically detect irreversible oxidized peroxiredoxin. The subtype of peroxiredoxin is not distinguished, and the active center is It refers to an antibody that distinguishes only those that are irreversibly oxidized. The antibody may be a polyclonal antibody or a monoclonal antibody, and antibody fragments such as Fab derived from a monoclonal antibody, reduced IgG derived from a monoclonal antibody, and Fab ′ antibody can also be used. Moreover, as long as an antibody can be applied to an immunoassay, it may be produced based on a known method, or a commercially available antibody may be used. For example, monoclonal antibodies that recognize irreversible oxidized peroxiredoxin subtypes 1 to 4 (Anti Peroxyredoxin-SO ₃ , LabFrontier, and Anti Peroxyredoxin-SO ₃ , AbCam) may be mentioned.

  Subsequently, the antigen-immobilized immunoassay method according to the present embodiment will be described. The antigen-immobilized immunoassay method of the present embodiment comprises a step of immobilizing an antigen in a body fluid collected from a subject, that is, an irreversible oxidized peroxiredoxin (antigen immobilization step), and an irreversible immobilized on the solid phase. A step of forming an immune complex of the oxidized oxidized peroxiredoxin and the first antibody (primary reaction step), a step of detecting the immune complex and measuring the abundance of the irreversible oxidized peroxiredoxin ( Measurement step).

  The antigen immobilization step can be performed as follows. For example, a solid phase such as a 96-well plate made of polystyrene is used as the solid phase, and a body fluid collected from the subject or a diluted solution thereof (for example, diluted with PBS or PBS containing a blocking agent) is added to 4 ° C to The antigen is fixed by allowing it to stand at room temperature overnight or at 37 ° C. for 1 to 3 hours to adsorb the antigen to the bottom of the well. Then, in order to prevent nonspecific adsorption of the antibody to the inner bottom surface of the well on which the sample is immobilized, blocking is performed at 4 ° C. to 37 ° C. for 1 to 3 hours using a solution containing a blocking agent such as BSA. I do. In the next primary reaction step, a solution in which the first antibody is diluted is added to a well (solid phase) in which irreversible oxidized peroxiredoxin is immobilized, and the solution is allowed to stand at 4 ° C. to room temperature for 1 to 3 hours. It is done by placing. In addition, reaction conditions, such as reaction temperature and reaction time, can be suitably optimized by those skilled in the art.

  Next, the indirect competitive immunoassay method according to this embodiment will be described. An indirect competitive immunoassay method according to an embodiment of the present invention includes a step of immobilizing a standard antigen, that is, a standard irreversible oxidized peroxiredoxin having a constant concentration (standard antigen immobilization step), and a body fluid collected from a subject. In the presence of irreversible oxidized peroxiredoxin, a process of forming an immune complex between the immobilized irreversible oxidized peroxiredoxin and the first antibody (primary reaction process), and detecting the immune complex And a step (measurement step) of measuring the abundance of irreversible oxidized peroxiredoxin in the body fluid collected from the subject.

  The indirect competitive immunoassay is a method that utilizes a solid-phased standard antigen and an antigen in a sample to form an immune complex with a first antibody in a competitive manner. The amount of the antigen present in the sample is calculated based on the signal intensity derived from the immunocomplex of the standard antibody immobilized on the first antibody and the first antibody after washing away the immune complex with the antibody. Is the method. As the solid phase, anti-irreversible oxidized peroxiredoxin antibody, blocking solution and the like used in the indirect competitive immunoassay method of the present embodiment, those described above can be used.

  The kit according to this embodiment includes a solid phase for performing the above evaluation method, a first antibody that specifically recognizes irreversible oxidized peroxiredoxin, and instructions for use. In one embodiment, The first antibody is labeled, and in other embodiments, further comprises a second molecule that specifically recognizes the first antibody, wherein the second molecule is a labeled molecule. Therefore, the measurement step of the antigen-immobilized immunoassay method, in one embodiment, detects the signal intensity derived from the label of the first antibody in the immune complex, and abundance of irreversible oxidized peroxiredoxin. In another embodiment, the method further comprises the step of forming a three-component immune complex (secondary reaction step) of the immune complex and the second molecule, And a step of measuring the abundance of irreversible oxidized peroxiredoxin (measurement step) by detecting the signal intensity derived from the label of the second molecule in the immune complex of the subject. Quantification is performed according to a conventional method, and the fatigue level is evaluated by the above-described fatigue level evaluation method.

  The label for measuring the abundance of irreversible oxidized peroxiredoxin in the immune complex is preferably an enzyme, a fluorescent substance, or a radioactive substance. Examples of the enzyme include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Examples of the fluorescent substance include fluorescein and its derivatives, Cy3, Cy5, Alexa Fluoro dye (Molecular Probes) and the like. A combination of the first antibody and the second molecule in other embodiments can be appropriately selected by those skilled in the art. For example, a combination of a primary antibody and a secondary antibody, a biotin-labeled primary antibody and avidin (Or streptavidin) combination, avidin (or streptavidin) labeled primary antibody and biotin combination, and the like. A combination using a sugar chain binding protein lectin may also be used.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not restrict | limited to a following example.

The abbreviations and names used in the present specification and examples are listed.
BPB: bromophenol blue DTT: dithiothreitol EBSS: Earl's Balanced Salt Solutions
EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum LC: liquid chromatography MEM: minimum essential medium MS: mass spectrometry MTT: methylthiazole tetrazolium PBS: phosphate buffered saline SDS: sodium dodecyl sulfate TFA: trifluoroacetic acid

(Example 1: Oxidative stress loading experiment using nerve cells)
(Experimental materials and culture conditions)
Mouse neuroblastoma (Neuro-2a) was obtained from the Human Science Research Resource Bank (IFO50081). All culture-related reagents and media were manufactured by Invitrogen (Gibco). The medium used for cell maintenance and passage is 10% FBS MEM containing 2 mM GlutaMax-1 and antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin), hereinafter referred to as maintenance medium.

The ampoule with neuroblastoma stored at −80 ° C. was rapidly thawed and transferred to a 15 ml tube pre-filled with 10 ml of maintenance medium. After centrifugation at 1000 rpm for 3 min, the supernatant was aspirated and 5 ml of fresh maintenance medium was added and pipetted. The obtained cell suspension was transferred to a 6 cm dish (Falcon) and immediately cultured in an incubator at 5% CO ₂ and 37 ° C. Cells were passaged 48 hours after the start of culture.

In order to maintain the cells normally, it takes about 2 days when the cells are passaged from a subconfluent state at a dilution ratio of 1/6, and about 5 days when they are passaged at a dilution ratio of 1/20 As a guideline, the cells were again grown to a subconfluent state. For reproducibility of experiments, when subcultured at a dilution ratio of 1/20, the number of culture days until the next subculture should be up to 5 days. The limit was 6-7 generations. A 75 cm ² flask (Asahi Techno Glass) was used for the main culture. The passage method is as follows. The maintenance medium was aspirated and rinsed with PBS, 2 ml of 0.25% trypsin-1 mM EDTA 4Na was added and left at room temperature for 5 minutes, and 5 ml of maintenance medium was added and pipetted. This cell suspension was diluted and passaged with fresh maintenance medium.

(Examination of oxidative stress load conditions)
For pre-culture of the oxidative stress load experiment, subconfluent cells were trypsinized and 2% FBS MEM (hereinafter referred to as “2% serum medium”) containing antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin). ), Diluted to an appropriate cell density, and plated on a culture dish. A 96-well plate coated with polylysine was used for the MTT assay (Example 2), and a 10 cm dish (Asahi Techno Glass) was used for ELISA analysis (Example 3) and two-dimensional electrophoresis analysis (Example 4). It was. The cell density was 100 μl / well at 1.0 × 10 ⁵ cells / ml in the case of a 96-well plate and 10 ml / dish at 2.5 × 10 ⁵ cells / ml in the case of a 10 cm dish.

  An oxidative stress loading experiment was conducted after 42 hours of pre-culture. The procedure is as follows. After removing the 2% serum medium by aspiration and rinsing the cells once with PBS, EBSS supplemented with hydrogen peroxide to various concentrations was added and cultured for 1 hour. Thereafter, the cells were rinsed twice with PBS, and after preparation of EBSS, sample preparation for use in MTT assay (Example 2) or two-dimensional electrophoresis analysis (Example 4) was started.

(Example 2: MTT assay)
Immediately after adding 100 μl of EBSS to each well, 11 μl of a 5 mg / ml MTT solution prepared by dissolving MTT (Sigma Aldrich) in EBSS was added, and cultured in a CO ₂ incubator for 4 hours. After aspirating the supernatant, 100 μl of DMSO was added, and MTT formazan was sufficiently dissolved by pipetting. The absorbance at 595 nm of the obtained lysate was measured with a microplate reader, and the result was displayed as% of the absorbance of the control (FIG. 1).

  Under these experimental conditions, the hydrogen peroxide concentration at which the MTT formazan production rate (MTT reducing ability) indicating cell activity is about 50% is 0.025 mM in the case of a 96-well plate, and 0.05 mM in the case of 10 cm dish. there were.

(Example 3: Effect of oxidative stress load on irreversible oxidized peroxiredoxin secretion in neurons)
Neuro-2a was cultured for 42 hours using a polylysine-coated 10 cm dish and 2% serum medium, and then the medium was changed to EBSS supplemented with 0.05 mM hydrogen peroxide, followed by treatment with hydrogen peroxide for 60 minutes. Thereafter, the cells were rinsed with PBS and then cultured in EBSS for 3 hours. The amount of irreversible oxidized peroxiredoxin in the culture supernatant was quantified by the antigen-immobilized ELISA method shown below.

Anti-irreversible oxidized peroxiredoxin antibodies include biotin-labeled monoclonal antibodies that recognize irreversible oxidized peroxiredoxin subtypes 1 to 4 (Anti Peroxyredoxin-SO ₃ AB [10A1], product number: ab16951, AbCam) Was used.

  First, a sample (medium supernatant) diluted with a coating buffer (0.05 M carbonate buffer) was added at 100 μl per well of a 96-well plate and allowed to stand at 4 ° C. overnight. Thereafter, 200 μl of a blocking solution (PBS containing 3% BSA) was added per well, and blocking was performed at room temperature for 2 hours.

Next, the well was washed with PBS containing 0.025% Tween 20 (hereinafter referred to as “washing solution”) to remove excess blocking solution. Subsequently, the anti-irreversible oxidized peroxiredoxin antibody labeled with biotin was diluted to 4 μg / ml in PBS containing 0.05% Tween 20 and 0.5% BSA (hereinafter referred to as “BT-PBS”). Then, 100 μl was added per well and reacted at room temperature for 2 hours. After the reaction, the inside of the well was washed with a washing solution, and horseradish peroxidase-labeled streptavidin (Nacalai Tesque) diluted 8000 times with BT-PBS was added at 100 μl per well and allowed to react at room temperature for 20 minutes. After washing, 100 μl of chromogenic substrate solution was added per well and allowed to react for 1 hour at room temperature. As the chromogenic substrate solution, a solution obtained by adding 10 μl of hydrogen peroxide solution immediately before use to 10 ml of citrate-phosphate buffer in which 25 mg of orthophenylenediamine was dissolved was used. The color development reaction was stopped by adding 100 μl of 2MH ₂ SO ₄ per well, and the absorbance at 492 nm was measured. A blank value was subtracted from the measured value of each well, a standard curve was obtained from the concentration and absorbance of the standard product, and the amount of irreversible oxidized peroxiredoxin in the sample was quantified.

  The results are shown in FIG. FIG. 2 is a graph showing the amount of irreversible oxidized peroxiredoxin secretion in cultured mouse neuroblastoma (Neuro-2a) under oxidative stress. The average value of the control (cultured with EBSS without hydrogen peroxide) was expressed as a relative value with the average value being 1 (mean ± SD). In the cell culture subjected to oxidative stress loading (0.05 mM hydrogen peroxide treatment, 60 minutes), the amount of irreversible oxidized peroxiredoxin secreted in the culture supernatant was 4 times that of the control. That is, it was found that the secretion of irreversible oxidized peroxiredoxin significantly increases depending on the oxidative stress load. Therefore, it is considered that measuring the amount of irreversible oxidized peroxiredoxin is effective as an index of oxidative stress that is a factor causing fatigue.

(Example 4: Proteome analysis by two-dimensional electrophoresis)
(Sample preparation)
The supernatant was removed from the 10 cm dish containing the subconfluent cells, and 10 ml of EBSS was added to each dish, followed by culturing in a CO ₂ incubator for 3 hours. The cultured EBSS medium collected gently was centrifuged at 1000 rpm for 3 min to remove slightly contaminated cells, and then used as a stock solution for a sample for two-dimensional electrophoresis. This stock solution was concentrated using an ultrafiltration membrane (Millipore) so that the protein concentration was 1 mg / ml, and desalted with a solution having the following composition to obtain a sample solution for two-dimensional electrophoresis. A commercially available product was used as the reagent.
urea 2.55g
Pharmalyte 3-10 0.1ml
DTT 0.05g
10% SDS 0.1ml
10% TritonX-100 1.0ml
Ultra pure water up to 5.0ml

(First dimension electrophoresis, isoelectric focusing)
As the dry strip gel, pH 4-7, 18 cm (Amersham) was used. The composition of the swelling liquid and the amount per gel were as follows, and these were prepared and used at the time of use.
urea 1.800g
thiourea 0.763g
DTT 0.010g
Pharmalyte 3-10 0.050ml
0.1M acetic acid 0.125ml
0.1% (w / v) Orange G 0.125 ml
20% (v / v) Triton X-100 0.500ml
Ultra pure water up to 5.0ml

  The swelling of the dried strip gel was performed by the following procedure. 5 ml of the swelling liquid per gel was put in a container (an acrylic tube with a silicone rubber stopper), the dried strip gel protective film was peeled off, and the gel surface was immersed in the swelling liquid. Using a mild mixer, the gel was swollen overnight at room temperature.

In the first dimensional electrophoresis (isoelectric focusing), energization was performed using the following program using an isoelectric focusing apparatus and a power supply manufactured by Anatech.
Step Voltage Time
1 500V 2h
2 700V 1h
3 1000V 1h
4 1500V 1h
5 2000V 1h
6 2500V 1h
7 3000V 1h
8 3500V 10h
9 500V keep

(SDS treatment and reduction alkalinization treatment)
The solutions used for the SDS treatment and the reduction alkalinization treatment were adjusted as follows.
SDS treatment solution (prepared at the time of use;
urea 1.813g
50% (v / v) glycerol 2.5ml
0.5 M Tris-HCl, pH 6.8, 0.25 ml
DTT 0.025g
0.1% (w / v) BPB 0.125 ml
10% (w / v) SDS 1.0 ml
Reduced alkylation solution (prepared when used, the following is the amount per bottle)
0.5 M Tris-HCl, pH 6.8, 0.25 ml
10% (w / v) SDS 1.0 ml
0.1% (w / v) BPB 0.125 ml
50% (v / v) glycerol 2.5ml
Ultrapure water 1.125ml
iodoacetamide 0.225g

  The SDS process was performed according to the following procedure. First, the gel strip after the first-dimensional electrophoresis was taken out with tweezers, rinsed with ultrapure water and poured with silicone oil. Next, the gel strip was placed in an acrylic tube containing the SDS treatment solution, stoppered with silicone rubber, and then incubated for 40 minutes using a mild mixer. The subsequent reductive alkylation procedure is as follows. The SDS-treated gel strip was placed in an acrylic tube containing a reduced alkylation treatment solution, and incubated for 20 minutes using a mild mixer. Immediately after the reductive alkylation treatment, the next operation, SDS electrophoresis, was started.

(Second dimension electrophoresis, SDS electrophoresis)
For the second dimensional electrophoresis (SDS electrophoresis), a second dimensional electrophoresis apparatus manufactured by Anatech and 7.5% acrylamide gel were used. SDS electrophoresis was performed by applying current for about 3.5 hours at a constant current of 40 mA per gel.

(Silver staining)
Silver staining was performed according to the instructions attached to the silver staining MS kit (Wako Pure Chemical Industries). The obtained gel electrophoresis image was taken with a CCD camera, and the data was stored in a computer.

  By performing the above operation, the influence of oxidative stress load on nerve cells was analyzed by two-dimensional electrophoresis. The resulting gel electrophoresis image is shown in FIG. As a result of analyzing the gel electrophoresis image, the spot No. in which the expression profile changes depending on the oxidative stress load. 1 and no. I found 2. Spot No. 1 and no. Both 2 had a molecular weight of approximately 22 kDa, and the isoelectric points (pI) were around 5.5 and 5.7, respectively. In contrast to the control, spot no. 1 appeared, and spot no. The protein corresponding to 2 was found to decrease.

(Example 5: Protein identification dependent on oxidative stress load by LC-MS / MS analysis)
(In-gel digestion process)
Spot No. obtained as a result of the analysis of Examples 1-3. 1 and no. In order to identify the protein corresponding to 2, LC-MS / MS analysis was performed by the following procedure. First, spot no. 1 and no. Each gel piece containing a protein corresponding to 2 was cut out. After desilvering these gel pieces, trypsin was added and treated at 37 ° C. for 20 hours to obtain sample solutions containing peptide fragments of the respective target proteins. This sample solution was submitted for LC-MS / MS analysis.

(LC-MS / MS analysis)
Analysis conditions in the LC-MS / MS analysis are as follows.
The HPLC apparatus used was Paradigm MS-4 (Michrom BioResearch, Inc., USA), and the HPLC column used was MAGIC C18 3μ 0.2 × 50 mm. Solvents used for HPLC were solvent A (2% acetonitrile / 0.1% formic acid, 2% acetonitrile / 0.1% TFA) and solvent B (90% acetonitrile / 0.1% formic acid, 90% acetonitrile / 0.1 % TFA), and the separation conditions were a flow rate of 1.0 μl / min and the following gradient conditions.
Gradient conditions: (min) Solvent B (%)
0.00 5.0
50.00 65.0
51.00 5.0
60.00 5.0

  As the mass spectrometer, LCQ Advantage (Thermo Finigan, USA) was used. The analysis was performed using Nanoflow-LC NSI as the ionization method, positive mode as the ionization mode, and a capillary voltage of 2 kV.

(Identification of target protein by database search)
The data obtained by LC-MS / MS analysis was compared with two types of protein databases of MASCOT and TurboSEQUEST, and the target protein was identified. As a result, spot no. 1 and no. The inventors have found that both proteins corresponding to 2 are peroxiredoxin 2 (calculated molecular weight is about 22 kDa). In combination with the isoelectric points of both proteins revealed from the results of Example 4 and the findings so far (Non-patent Document 1), spot No. The protein corresponding to 2 is reduced peroxiredoxin 2 and spot no. The protein corresponding to 1 was found to be peroxiredoxin 2 (irreversible oxidized peroxiredoxin 2) in which the 51 Cys thiol group of peroxiredoxin 2 was sulfonated or sulfinated.

  From the above results, it was confirmed that the amount of irreversible oxidized peroxiredoxin secreted increased significantly depending on the oxidative stress load, consistent with the results of Example 3. It was also found that the ratio of the amount of irreversible oxidized peroxiredoxin and reduced peroxiredoxin varies depending on the oxidative stress load. Therefore, in addition to measuring the amount of irreversible oxidized peroxiredoxin, measuring the ratio of the amount of irreversible oxidized peroxiredoxin to the amount of peroxiredoxin is the factor that causes fatigue. It is considered effective as an index of stress.

(Example 6: Construction of plasma irreversible oxidized peroxiredoxin quantification method by indirect competitive ELISA)
In measuring irreversible oxidized peroxiredoxin in plasma containing a large amount of contaminating proteins, a new indirect competitive ELISA method is used as a more sensitive measurement method than the antigen sample solid-phase ELISA method shown in Example 3. The measurement method by was constructed. Details of the method are described below.

As the anti-irreversible oxidized peroxiredoxin antibody, a biotin-labeled polyclonal antibody that recognizes irreversible oxidized peroxiredoxin subtypes 1 to 4 (Anti Peroxyredoxin-SO ₃ AB, product number: ab16830, AbCam) was used. .

  First, prepare a standard irreversible oxidized peroxiredoxin standard diluted in coating buffer (0.05 M carbonate buffer), add 100 μl per well of a 96-well plate for ELISA, and allow to stand at 4 ° C. overnight. I put it. After washing the inside of the well three times with a washing solution, a blocking solution (PBS containing 3% BSA) was added at 300 μl per well, and blocking was performed at room temperature for 2 hours. In addition, as a irreversible oxidized peroxiredoxin standard product, a 5-fold diluted solution of Neuro-2a culture supernatant treated with hydrogen peroxide was used.

Next, the inside of the well was washed with a washing solution three times to remove excess blocking solution. Subsequently, irreversible oxidized peroxiredoxin for standard curve dilution diluted with BT-PBS (PBS containing 0.05% Tween 20 and 0.5% BSA) (or a reference plasma sample serially diluted with BT-PBS) Alternatively, a plasma sample for measurement diluted with BT-PBS (50 μl / 1 well) and a biotin-labeled anti-Prx-SO ₃ polyclonal antibody diluted with BT-PBS (2 μg / ml, 50 μl / 1 well) are added. And allowed to react at room temperature for 2 hours. After the reaction, the inside of the well was washed with a washing solution, and horseradish peroxidase-labeled streptavidin (Nacalai Tesque) diluted 8000 times with BT-PBS was added at 100 μl per well and allowed to react at room temperature for 20 minutes. After washing, 100 μl of chromogenic substrate solution was added per well and allowed to react for 1 hour at room temperature. As the chromogenic substrate solution, one prepared in the same manner as in Example 3 was used. The color development reaction was stopped by adding 100 μl of 2MH ₂ SO ₄ per well, and the absorbance at 492 nm was measured. A blank value was subtracted from the measured value of each well, a standard curve was obtained from the concentration and absorbance of the standard product, and the amount of irreversible oxidized peroxiredoxin in the sample was quantified.

(Example 7: Measurement of irreversible oxidized peroxiredoxin concentration in plasma in fatigue model animals)
Fatigue model rats were prepared according to the method of M. Tanaka et al. (Neurosci. Lett. (2003) vol. 352, p. 59-162). This is a model that inhibits deep sleep and is also said to be an overwork death model. This model is created by submerging 7-week-old male SD rats for 5 days (water depth 1.5 cm). Since this model is accompanied by a decrease in body weight, in addition to a normal rearing group as a control, a group was also provided that was close to the weight change of the fatigue group by dietary restriction (7.5 g / day) (FIG. 4). Five days after the start of water immersion, blood was collected from the posterior vena cava using heparin to obtain plasma.

  About the said rat group, the measurement result of the plasma irreversible oxidation type peroxiredoxin density | concentration by indirect competition ELISA method is shown (FIG. 5). Compared with the control group, the plasma irreversible oxidized peroxiredoxin concentration was significantly higher in the water immersion group. There was no significant difference between the control group and the diet restriction group.

  From the above, it was found that the concentration of irreversible oxidized peroxiredoxin was significantly increased in the plasma of fatigued rats. It was also confirmed that the increase in the concentration of irreversible oxidized peroxiredoxin in plasma due to fatigue could be measured. Therefore, measuring the irreversible oxidized peroxiredoxin concentration is considered to be effective as a method for objectively evaluating physical or mental fatigue from body fluids such as plasma.

Claims (8)

Measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from a subject;
Determining fatigue based on the amount of irreversible oxidized peroxiredoxin,
A method for evaluating fatigue level. Measuring the ratio of the amount of irreversible oxidized peroxiredoxin present in the body fluid collected from the subject to the amount of peroxiredoxin present;
Determining the degree of fatigue based on the ratio between the amount of irreversible oxidized peroxiredoxin and the amount of peroxiredoxin;
A method for evaluating fatigue level. The step of measuring the abundance of the irreversible oxidized peroxiredoxin comprises the following steps:
Immobilizing irreversible oxidized peroxiredoxin in body fluid collected from a subject; and
A step of forming an immune complex between a solid-phased irreversible oxidized peroxiredoxin and a labeled first antibody, wherein the first antibody is specific to the irreversible oxidized peroxiredoxin Recognizing and labeling the antibody,
Detecting the signal intensity derived from the label of the first antibody in the immune complex and measuring the abundance of irreversible oxidized peroxiredoxin;
The fatigue degree evaluation method according to claim 1, comprising: The step of measuring the abundance of the irreversible oxidized peroxiredoxin comprises the following steps:
Immobilizing irreversible oxidized peroxiredoxin in body fluid collected from a subject; and
A step of forming an immune complex between a solid-phased irreversible oxidized peroxiredoxin and a first antibody, wherein the first antibody specifically recognizes the irreversible oxidized peroxiredoxin A process that is an antibody to
Forming a ternary immune complex of the immune complex and a second molecule, wherein the second molecule is a molecule that specifically recognizes and labels the first antibody Process,
Detecting the signal intensity derived from the label of the second molecule in the tripartite immune complex and measuring the abundance of irreversible oxidized peroxiredoxin;
A method for evaluating the degree of fatigue according to claim 1 or 2. The step of measuring the abundance of the irreversible oxidized peroxiredoxin comprises the following steps:
Immobilizing standard irreversible oxidized peroxiredoxin; and
In the step of forming an immune complex between the solidified standard irreversible oxidized peroxiredoxin and the labeled first antibody in the presence of the irreversible oxidized peroxiredoxin in the body fluid collected from the subject The first antibody is a labeled antibody that specifically recognizes irreversible oxidized peroxiredoxin; and
Detecting the signal intensity derived from the label of the first antibody in the immune complex, and measuring the abundance of irreversible oxidized peroxiredoxin in a body fluid collected from the subject;
The fatigue degree evaluation method according to claim 1, comprising: The step of measuring the abundance of the irreversible oxidized peroxiredoxin comprises the following steps:
Immobilizing standard irreversible oxidized peroxiredoxin; and
In the step of forming an immune complex between the solidified standard irreversible oxidized peroxiredoxin and the labeled first antibody in the presence of the irreversible oxidized peroxiredoxin in the body fluid collected from the subject The first antibody is a labeled antibody that specifically recognizes irreversible oxidized peroxiredoxin; and
Forming a ternary immune complex of the immune complex and a second molecule, wherein the second molecule is a molecule that specifically recognizes and labels the first antibody Process,
Detecting the signal intensity derived from the label of the second molecule in the tripartite immune complex and measuring the abundance of irreversible oxidized peroxiredoxin in the body fluid collected from the subject;
A method for evaluating the degree of fatigue according to claim 1 or 2.   A kit for evaluating the degree of fatigue, comprising a solid phase for performing the evaluation method according to claim 3 or 5, a first antibody, and instructions for use.   A kit for evaluating the degree of fatigue, comprising a solid phase, a first antibody, a second molecule, and instructions for use for performing the evaluation method according to claim 4 or 6.