JP5116485B2 - Stress determination method, stress determination marker, diagnostic agent for stress determination, and stress determination system - Google Patents

Description

  The present invention relates to a stress determination method, a stress determination marker, a stress determination diagnostic agent, and a stress determination system.

  In recent years, diseases caused by mental stress have become a social problem, and development of a method for simply and objectively diagnosing the mental state of humans and non-human animals is required. In such a stress society, development of functional foods that are effective in anti-fatigue and anti-stress is required.

  At present, since there is no highly reliable marker for stress determination and no simple and objective stress determination method, the determination of stress requires a complicated examination by a psychiatrist. Moreover, the development of functional foods is currently stagnant because stress-reducing foods are not approved as foods for specified health use for the above reasons.

  Therefore, in order to overcome such a current situation, searching for a marker for stress determination is performed. As such markers, heart rate and blood pressure, and concentrations of corticosteroids and neurotransmitters in blood are known. Recently, cortisol, chromogranin A, IgA, and amylase in saliva are used as stress determination markers. It is proposed to be available.

JP 2000-275248 A JP-A-11-326318

  However, since heart rate and blood pressure are transient phenomena, they are affected by the timing of measuring stress, and it is clear that the components in the blood are affected by the stress caused by the needle stick at the time of blood collection. is there. As for the components in saliva, it is known that cortisol has a slow response to stress and is influenced by the feedback mechanism, and chromogranin A, IgA and amylase gradually reveal that stress is not accurately reflected. It is becoming.

  Therefore, an object of the present invention is to provide a method capable of easily and objectively determining stress. Another object of the present invention is to provide a stress determination marker, a stress determination diagnostic agent, and a stress determination system that can be applied to such a method.

  The stress determination method of the present invention is characterized in that stress is determined using the concentration of Zn-α2-glycoprotein in the body fluid of a test animal as an index.

  Since the method of the present invention uses the concentration of Zn-α2-glycoprotein in the body fluid of the test animal as an indicator, the degree of stress received by the test animal can be detected with high sensitivity, and the stress can be easily and objectively applied. Can be determined. Moreover, it can be implemented in the home because of its simplicity, and is useful for collecting information for developing functional foods that are effective in anti-fatigue and anti-stress.

  In the above method, the increase and decrease in the concentration of Zn-α2-glycoprotein can be determined as the increase and decrease in stress, respectively. That is, when the concentration of Zn-α2-glycoprotein in the body fluid of the test animal before and after the work for which the stress is to be examined is measured, and when this concentration increases, it is determined that the stress has increased, and this concentration has decreased It can be determined that the stress has decreased.

  The stress determination method of the present invention includes a measurement step for measuring the concentration of Zn-α2-glycoprotein in the body fluid of a test animal, the concentration of Zn-α2-glycoprotein obtained in the measurement step, and a body fluid at rest Comparing the concentration of Zn-α2-glycoprotein in the solution and the concentration of Zn-α2-glycoprotein obtained in the measurement step above the concentration of Zn-α2-glycoprotein in the body fluid at rest And a determination step of determining that stress has occurred when the value is high.

  Once the concentration of Zn-α2-glycoprotein in the body fluid at the time of rest of the test animal is measured as a reference value, the concentration of Zn-α2-glycoprotein in the body fluid is measured when it is desired to determine stress, By comparing with the reference value, the degree of stress received by the test animal can be easily and objectively determined.

  The body fluid is preferably a body fluid collected non-invasively, and saliva is particularly preferable as the body fluid.

  The above stress determination method can be determined using the blood of the test animal, but the blood sampling action can only be performed by qualified personnel such as doctors, and because only the act of sticking the injection needle causes stress, It is preferable to determine stress using body fluids collected non-invasively such as saliva, sweat, and tears. The concentration of Zn-α2-glycoprotein increases in the above-mentioned non-invasively collected body fluid according to the degree of stress experienced by the test animal. Simple and objective determination is possible.

  By using saliva as a non-invasively collected body fluid, a necessary amount can be easily collected regardless of the collection time. Therefore, the degree of stress received by the test animal can be determined more simply and objectively.

  The marker for stress determination of the present invention is characterized by comprising Zn-α2-glycoprotein.

  Since the concentration of Zn-α2-glycoprotein present in the body fluid of the test animal rises with high sensitivity within a few minutes when subjected to stress, it can be used as a stress determination marker.

  The diagnostic agent for stress determination of the present invention is characterized by containing an anti-Zn-α2-glycoprotein antibody.

  The anti-Zn-α2-glycoprotein antibody contained in the diagnostic agent for stress determination specifically binds to the Zn-α2-glycoprotein present in the body fluid of the test animal, and the Zn-α2-glycoprotein in the body fluid Since it can be quantified, the diagnostic agent can be used to easily and objectively determine the degree of stress applied to the subject animal.

  The present invention also provides a receiving means for receiving information on the concentration of Zn-α2-glycoprotein in the body fluid of a test animal transmitted from the terminal, and the concentration of Zn-α2-glycoprotein received by the receiving means. The stress information storage means for storing the information of the stress, the stress determination means for determining the stress using the Zn-α2-glycoprotein concentration information stored by the stress information storage means as an index, and the stress determination means A stress determination system including a determination result transmission unit that transmits a determination result to a terminal is provided.

  The stress determination system can be connected to a telecommunications network in which the terminal and the medical related information providing system can be connected, and the medical related information providing system determines the health condition of the test animal based on the received determination information. If it has the means to determine the medical relevant information regarding this, and the means to transmit this medical relevant information to a terminal, the information of the density | concentration of Zn- (alpha) 2-glycoprotein of the bodily fluid of a test animal will be transmitted to the said stress judgment system By doing so, information useful for relieving stress can be obtained from a portable terminal or an externally installed terminal when necessary. Thereby, accumulation of further stress can be prevented, or sleep time can be controlled according to the degree of stress. Furthermore, if the information provided by the medical related information providing system to the subject is devised, more detailed information such as optimal meals, beverages, and medicines can be provided.

  The stress determination method of the present invention makes it possible to determine stress easily and objectively. Further, compared to a stress determination method using cortisol and chromogranin A, which are known stress determination markers, there is less variation between individuals, and the degree of stress can be determined with high sensitivity.

  The stress determination method of the present invention can use not only blood of a test animal but also body fluid collected non-invasively such as saliva, and the degree of stress received by the test animal can be simply and objectively measured at home. Can be determined.

  The marker for stress determination and the diagnostic agent for stress determination of the present invention can be used for simply and objectively determining the degree of stress received by a test animal.

  The stress judgment system of the present invention can obtain information useful for relieving stress from a portable terminal or an externally installed terminal when necessary. Thereby, accumulation of further stress can be prevented, or sleep time can be controlled according to the degree of stress.

It is a block diagram for demonstrating the stress judgment system which concerns on embodiment. It is the figure which showed the time of collection of the time of the Kraepelin test employ | adopted as a stress load test, and a saliva sample. It is the figure which showed the change of the density | concentration of Zn- (alpha) 2- glycoprotein in the saliva of the test subject 1 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2- glycoprotein in the saliva of the test subject 2 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 3 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2- glycoprotein in the saliva of the test subject 4 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 5 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of the Zn- (alpha) 2- glycoprotein in the saliva of the test subject 6 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 7 before and after a stress load test (Kraepelin work). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 8 before and behind a stress load test (Advanced TrailMaking Test; ATMT). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 9 before and behind a stress load test (Advanced Trail Making Test; ATMT). It is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subject 10 before and behind a stress load test (Advanced Trail Making Test; ATMT).

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Stress judgment system, 12 ... Reception means, 14 ... Stress information storage means, 16 ... Stress judgment means, 18 ... Determination result transmission means, 20 ... Network, 30 ... Terminal 40, medical related information providing system 42 receiving means 44 determination information storing means 46 medical information determining means 48 48 determination result transmitting means

  Hereinafter, preferred embodiments of the present invention will be described in detail.

(Stress judgment method)
The term “stress” in the present invention refers to distortion or modulation that is shown to the body by some kind of stimulus (stresser). “Stress” includes heavy pressure, pressure, pressure, strain, and tension, and such stimuli and situations are mentally and physically burdensome.

  “Stressors” are: 1) physical stressors represented by stimuli such as temperature and noise, 2) chemical stressors represented by oxygen deficiency / abundance, chemical damage, nutritional deficiencies, etc., 3) diseases caused by pathogenic bacteria, etc. It can be divided into four types: biological stressors represented by 4) mental stressors represented by troubles of human relations, mental pain, anger, anxiety, hatred, and tension. In addition, it is said that most of the causes of annoying people who feel "stress builds up" are due to the mental stressor of 4).

  Examples of the “test animal” include humans and non-human animals, and humans, pets and livestock are preferable. Examples include monkeys, dogs, cats, mice, rats, guinea pigs, birds, cows, horses, sheep and rabbits. It can be illustrated.

  “Body fluid” includes blood, lymph fluid, tissue fluid, cerebrospinal fluid, synovial fluid, mucus, hormones, digestive fluid, saliva, tears, aqueous humor, sweat, urine, etc., and the stress determination method of the present invention. As the body fluid to be used, blood, saliva, tears, aqueous humor, sweat and urine are preferable, and saliva is more preferable.

  The “non-invasively collected bodily fluid” means a bodily fluid collected from a test animal without pain or bleeding, and does not include bodily fluids collected by inserting an injection needle or the like into the test animal. Therefore, saliva, tears, aqueous humor, sweat, urine, and the like correspond to the body fluids.

  “Zn-α2-glycoprotein” is a kind of plasma protein found in blood, and its function has been conventionally unknown, but the present invention has revealed the function as a marker for stress determination. Human Zn-α2-glycoprotein is a glycoprotein consisting of 298 amino acids and having 3 N-linked sugar chains (SEQ ID NO: 1; Accession No. AAH33830), and the base sequence of this gene is registered with GenBank. (SEQ ID NO: 2; Accession No. BC033830). Zn-α2-glycoprotein is known to have a three-dimensional structure similar to that of MHC class I protein and to easily precipitate when zinc ions are added and to have RNase activity. In addition, Zn-α2-glycoprotein is abbreviated as ZAG among those skilled in the art.

  Next, a stress determination method according to the first embodiment of the present invention will be described.

  The stress determination method according to the first embodiment measures the concentration of Zn-α2-glycoprotein in the body fluid of the subject animal before and after the work for which the stress is to be examined, and the stress increases when this concentration increases. This is a method for judging that the stress is lowered when the concentration is lowered.

  Here, the “work to investigate stress” means a work that is mentally or physically burdened, for example, work, study, sports and work that constitutes a part thereof, and under high temperature, low temperature and noise. Work, painful work, etc.

  In the determination of stress, the concentration of Zn-α2-glycoprotein in the body fluid of the subject animal before and after the work for which stress is to be examined is compared, and it is determined that the stress has increased when the concentration after the work has increased. The degree of stress can be determined by setting a separate standard. For example, when the concentration of Zn-α2-glycoprotein is less than 2 times, it can be judged as low stress, when it is 2 times or more and less than 3 times, medium stress, and when it is 3 times or more, it can be judged as high stress.

  Next, a stress determination method according to the second embodiment of the present invention will be described.

  The stress determination method according to the second embodiment includes a measurement step of measuring the concentration of Zn-α2-glycoprotein in the body fluid of a test animal, and the concentration and rest of the Zn-α2-glycoprotein obtained in the measurement step. The comparison step of comparing the concentration of Zn-α2-glycoprotein in body fluid at the time and the concentration of Zn-α2-glycoprotein obtained in the measurement step is the Zn-α2-glycoprotein in the body fluid at rest And a determination step of determining that stress has occurred when the concentration is higher than the concentration of.

  Here, “resting” refers to the time when the test animal is not stressed, and is usually most resting when sleeping, but immediately after waking up because it is difficult to collect bodily fluids. (As soon as you get up) is preferred. However, since the degree of stress can be relatively determined, any time point at which no stress is considered can be determined as resting.

  In the measurement process, body fluid is collected when it is desired to determine the stress, and the concentration of Zn-α2-glycoprotein in the body fluid is measured. Any method can be used as long as the target protein can be quantified. Also good. For example, ELISA method, Western blotting method, image analysis of stained region (spot) obtained by staining protein fractionated by two-dimensional electrophoresis, analysis by protein chip, quantitative analysis by affinity chromatography, etc. Analysis by ELISA and protein chip is preferred.

  In the comparison step, the concentration of Zn-α2-glycoprotein measured in the measurement step is compared with the concentration of Zn-α2-glycoprotein in the body fluid at rest. For example, obtaining the difference between the concentration of Zn-α2-glycoprotein obtained in the measurement step and the concentration of Zn-α2-glycoprotein at rest, or examining the value obtained by dividing one concentration by the other It is done. The concentration can be obtained by ELISA, protein chip analysis, quantitative analysis by affinity chromatography, or the like. In addition, a stained region (spot) obtained by staining the protein fractionated by two-dimensional electrophoresis is imaged, and the degree of staining per unit area is digitized (for example, pixel intensity). Can be made the concentration of Zn-α2-glycoprotein (however, there is no unit).

  In the determination step, it is determined that stress has occurred when the concentration of Zn-α2-glycoprotein obtained in the measurement step is higher than the concentration of Zn-α2-glycoprotein in the body fluid at rest. Can be determined by setting a separate standard. For example, when the concentration of Zn-α2-glycoprotein is less than 2 times, it can be judged as low stress, when it is 2 times or more and less than 3 times, medium stress, and when it is 3 times or more, it can be judged as high stress.

(Marker for stress determination)
The stress determination marker of the present invention is composed of Zn-α2-glycoprotein. Here, the “stress determination marker” refers to an index for determining stress, and when stress occurs. If it can be changed and its degree can be displayed, it corresponds to a stress determination marker. As stress determination markers, heart rate, blood pressure, corticol and other corticosteroids such as test animals, neurotransmitters such as adrenaline and noradrenaline, chromogranin A, IgA, amylase and the like have been known. Regarding α2-glycoprotein, since its function has been unknown, it has not been known at all whether it can be used as a “stress determination marker”.

  This Zn-α2-glycoprotein is present in body fluids of test animals and can be quantified by various immunological techniques using anti-Zn-α2-glycoprotein antibodies. For example, Zn-α2-glycoprotein contained in saliva or blood can be quantified using techniques such as ELISA, Western blotting, analysis by protein chip, quantitative analysis by affinity chromatography, etc., but by ELISA method and protein chip Analysis is preferred.

(Diagnostic agent for stress determination)
By containing an anti-Zn-α2-glycoprotein antibody, a diagnostic agent for stress determination can be provided.

  “Diagnostic agent for stress determination” refers to a diagnostic agent for determining stress, and includes diagnostic agents containing proteins, nucleic acids, low molecular weight compounds, etc. that can specifically and quantitatively detect the above-mentioned stress determination markers. To do.

  The diagnostic agent for stress determination of the present invention contains an anti-Zn-α2-glycoprotein antibody, but if it does not inhibit the action of specifically recognizing the antigen of this antibody, other buffers, protease inhibitors, An SH group protecting agent or the like may be contained. The anti-Zn-α2-glycoprotein antibody may be an antibody that specifically recognizes the Zn-α2-glycoprotein of the test animal, and may be either a polyclonal antibody or a monoclonal antibody. Polyclonal antibodies can be prepared by immunizing rabbits with Zn-α2-glycoprotein of a test animal or a synthetic peptide thereof, and monoclonal antibodies can be prepared by the hybridoma method.

  The diagnostic agent for stress determination of the present invention can be used for detection and quantification of Zn-α2-glycoprotein contained in a body fluid collected from a test animal. The quantification can be performed, for example, by ELISA, Western blotting, analysis using a protein chip, or the like. As the quantification method, analysis by ELISA or protein chip is preferable.

(Application of stress determination method, stress determination marker and stress determination diagnostic agent)
By using the stress determination marker or the stress determination diagnostic agent of the present invention, a stress determination (determination) system for realizing the stress determination method of the present invention can be provided.

  FIG. 1 is a block diagram for explaining a stress determination system 10 according to the embodiment. The stress determination system 10 is physically a computer system (for example, a workstation or a personal computer) including a CPU (central processing unit), a memory, an input device such as a mouse or a keyboard, a display device such as a display, a storage device such as a hard disk. It is configured as. The stress determination system 10 includes, as functional elements, receiving means 12 (receiving unit) that receives information on Zn-α2-glycoprotein concentration (hereinafter referred to as “stress information”), and stress information that stores the stress information. A storage unit 14 (stress information storage unit), a stress determination unit 16 (stress determination unit) that determines the degree of stress based on the stored stress information, and a determination result transmission unit 18 (determination result transmission) that transmits this determination result Part).

  The stress determination system 10 can be connected to a network 20 (for example, a telecommunication network such as the Internet). Further, a terminal 30 and a medical related information providing system 40 can be connected to the network 20.

  Here, the terminal 30 is used as the terminal of the subject animal owner or the subject, and the medical related information providing system 40 receives the determination information transmitted from the determination result transmitting means 18 of the stress determination system 10 (receiving means 42 ( Receiving section), determination information storage means 44 (determination storage section) for storing this determination information, and medical information determination means 46 for determining medical-related information suitable for improving the health condition based on the stored determination information. (Medical information determination unit) and determination result transmission means 48 (determination result transmission unit) for transmitting this medical information to the terminal 30 are configured.

  Information (stress information) on the Zn-α2-glycoprotein concentration of the body fluid of the subject or the like transmitted from the terminal 30 of the subject animal owner or the subject (hereinafter referred to as “subject or the like”) is transmitted via the network 20. The information is received by the receiving means 12 (receiving section) of the stress judging system 10 and stored in the stress information storing means 14 (stress information storing section), and the stress judging means 16 (stress judging section) is based on the information. ) To determine the stress state.

  This determination result is transmitted by the determination result transmission unit 18 (determination result transmission unit), and is received by the reception unit 42 (reception unit) of the medical related information providing system 40 via the network 20, which stores the determination information. It is stored in the means 44 (determination storage unit). The medical information determination unit 46 (medical information determination unit) refers to the determination information stored in the determination information storage unit 44 (determination storage unit), and is related to medical treatment such as medicines, foods and beverages suitable for improving the health condition. Determine information. Then, the determined medical related information is transmitted by the determination result transmitting unit 48 (determination result transmitting unit), and is delivered to the terminal 30 such as a subject via the network 20.

  The stress determination system 10 may be implemented by a diagnostic institution, and the medical related information providing system 40 may be implemented by a health information providing organization such as a medical institution, a health food related company, a pharmaceutical company, a restaurant, a convenience store, or the like. . In addition, in order to improve the quality of information provided to subjects, information transmitted from the terminal 30 includes not only stress information but also information such as the age and sex of the body fluid provider. Is preferred. The information provided by the medical-related information providing system 40 to the subject etc. includes information on optimal meals and inappropriate meals, and commercials introduced under contracts with food, beverage and pharmaceutical manufacturers, etc. can do.

  The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

(Example 1) Stress load test and saliva collection:
As a stress load test, a Kraepelin test and an Advanced Trail Making Test (hereinafter, ATMT) were performed.

  The Kraepelin test is commonly used as a stress load test that gives mental stress, and is a written test that allows a subject to continuously perform mental arithmetic (Kreperin work) by adding a single digit. FIG. 2 is a diagram showing the time of the Kraepelin test employed as a stress load test and the time of collection of the saliva sample. The pre-stress saliva sample was collected before the Kraepelin work, and the post-stress saliva sample was taken after the Kraepelin work for 15 minutes. The Kraepelin operation was continued for 15 minutes thereafter. The reason for collecting the saliva sample after stress was 15 minutes after the Kraepelin operation was that the concentration of cortisol in the saliva decreased after the Creperin operation was performed for 30 minutes due to the feedback effect on the accumulation of cortisol in the saliva. Because there is a case to do. Furthermore, when the Kraepelin work is finished, the subject may be relieved to relieve stress.

  On the other hand, ATMT is a general test for evaluating the aging phenomenon of the brain, etc., and is a test in which numbers scattered irregularly on a personal computer screen are pushed as soon as possible in ascending order of numbers (Washio, Shinyaku and Clinical, 2000, 49, No. 4, p. 104). Specifically, first, the test subject is caused to press numbers 1 to 25 that are randomly scattered on the personal computer screen in the order of small numbers, and then the numbers 1 to 25 whose positions change randomly each time the numbers are pressed. The work is stressed by pressing the work in ascending order and performing 15 sets of this work continuously. The pre-stress saliva sample was collected before ATMT, and the post-stress saliva sample was collected immediately after ATMT.

(Example 2) Two-dimensional electrophoresis, image analysis and peptide mass finger printing analysis:
Each saliva sample was centrifuged at 3000 rpm for 10 minutes to remove solids, and the protein concentration was measured with a protein assay kit (Bio-Rad). Thereafter, each saliva sample equivalent to 0.1 mg of protein is transferred to a microtube, and the protein is precipitated by adding an equal amount of acetone. After centrifugation, a solubilized solution (9 M urea, 2% CHAPS is added to the resulting precipitate. 1% DTT, 0.3% (v / v) BioLyte 3-10) was added and dissolved. Each solubilized saliva sample was added to an IPG ReadyStripe gel (17 cm, pH 3-10NL; Bio-Rad), and the strip was swollen for 12 hours, followed by first-dimension isoelectric focusing, followed by 10 Second-dimensional electrophoresis was performed on a -16% gradient SDS-PAGE gel.

  After electrophoresis, the gel is stained with SYPRO RUBY protein gel stain (Molecular Probe), and image analysis is performed. The amount of expression between the saliva sample before stress (before Kraepelin operation) and after stress (after Kraepelin operation) Stained areas (spots) with different values were searched. The spot where the protein expression level was increased in the saliva sample after stress was cut out from the gel, treated with protease, then the precise mass of the fragment peptide was measured by MALDI-TOF MS, and Peptide Mass Fingerprinting (PMF) analysis was performed.

  As a result, the protein whose expression level was significantly increased before and after stress was Zn-α2-glycoprotein.

(Example 3) Measurement of stress determination marker protein concentration:
Each saliva sample collected before and after the Kraepelin operation was centrifuged at 3000 rpm for 10 minutes to remove solids, and the supernatant was used for measuring the concentration of the marker protein for stress determination. The concentration of cortisol in saliva was measured using a Cortisol ELISA kit (R & D systems), and the concentration of chromogranin A in saliva was measured using a chromogranin A ELISA kit (Yanaihara Laboratory).

  The concentration of Zn-α2-glycoprotein in saliva was determined by quantifying the degree of staining of the Zn-α2-glycoprotein spot by staining the gel after two-dimensional electrophoresis and analyzing the image. Specifically, first, each of the above saliva samples was fractionated by two-dimensional electrophoresis under the above conditions, and the proteins in the gel after electrophoresis were stained with SYPRO RUBY protein gel stain (Molecular Probe). . Thereafter, the stained gel image is taken into Molecular Imager FX (Bio-Rad), and the degree of staining per unit area is quantified with the image analysis software Melanie 3 (Bio-Rad) (pixel intensity). Were summed for the entire Zn-α2-glycoprotein spot to give the concentration of Zn-α2-glycoprotein (however, there are no units).

(Example 4) Comparison of the concentrations of cortisol, chromogranin A and Zn-α2-glycoprotein in saliva before and after the stress load test:
Four healthy individuals are arbitrarily selected and subjected to the stress load test (Kraepelin test) described above, before and after stress (before Creperin work) and after stress (after Creperin work) salivary cortisol, chromogranin A and Zn-α2 -The concentration of glycoprotein was measured by the above-described concentration measuring method. The measurement results are shown in Table 1.

  As a result, the concentration of cortisol in saliva after stress of subject 1 tended to increase significantly compared to that before stress, but subject 1.3 increased by about 1.3 times, and subjects 3 and 4 Then there was only a slight increase. Regarding the concentration of chromogranin A in the saliva after stress, a tendency to increase was observed in all of the subjects 1 to 4 compared to before stress, but it was about 1.1 to 1.6 times. It was.

  FIGS. 3-6 is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the subjects 1-4 before and after a stress load test (Kraepelin work) for every subject, respectively. As a result, the concentration of Zn-α2-glycoprotein in saliva after stress is about 3.1 times (Fig. 3) for subject 1 and about 2.4 times for subject 2 (Fig. 4) compared to before stress. In all cases, the increase was more than twice. Although it was about 1.3 times (Fig. 5) in subject 3 and about 1.1 times (Fig. 6) in subject 4, Zn-α2-glycoprotein in saliva was expressed in comparison with cortisol and chromogranin A. The amount was large and could be easily detected and determined.

  As described above, Zn-α2-glycoprotein significantly increases in response to stress than cortisol and chromogranin A, which are known as stress determination markers, and is difficult to determine when cortisol and chromogranin A are used as indices. Even in the case (Subjects 3 and 4), it was found that the determination of stress was possible.

(Example 5) Comparison of the concentration of Zn-α2-glycoprotein in saliva before and after the Kraperin work and ATMT by the ELISA method:
Comparison of the concentration of Zn-α2-glycoprotein in saliva before and after Kraperin work and ATMT in an ELISA system using a polyclonal antibody that specifically recognizes human Zn-α2-glycoprotein (hereinafter referred to as anti-ZAG antibody) did.

  An anti-ZAG antibody that specifically recognizes the human Zn-α2-glycoprotein shown in SEQ ID NO: 1 in the Sequence Listing is disclosed in the 12th Seminar of the New Chemistry Experiment Laboratory (Japan Biochemical Society, 1992, p. 1-12). Prepared according to the method described. Further, each saliva sample collected before and after the Kraepelin operation and ATMT was centrifuged at 3000 rpm for 10 minutes to remove the solid content, and the supernatant was used.

Quantification of Zn-α2-glycoprotein by ELISA using an anti-ZAG antibody was performed according to the following procedure. First, 100 μL of anti-ZAG antibody prepared to 25 μg / mL was added to each well of a 96-well plate and allowed to stand at 4 ° C. overnight to solid phase. Thereafter, each well was washed with a washing buffer (20 mM Tris-HCl, pH
8.0, 0.15 M NaCl, 0.05% Tween 20), and added blocking solution (1% BSA, 15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ ) to each well, incubated for 2 hours, and again Each well was washed 5 times with wash buffer. Thereafter, each saliva sample collected before and after the Kraperin work and ATMT and a sample of Zn-α2-glycoprotein with a known concentration were added to each well in an amount of 100 μL, and allowed to react with the anti-ZAG antibody for 2 hours. After washing 5 times with the washing buffer, 100 μL of anti-ZAG antibody (100 μg / mL) conjugated with peroxidase as a secondary antibody was added to each well and reacted at room temperature for 2 hours. Thereafter, each well was washed five times with a washing buffer, and 100 μL of a substrate solution (Sure Blue; KPL) was added to cause color development. After the color development sufficiently progressed, 100 μL of 1M hydrochloric acid was added to each well to stop the reaction, and the absorbance at 450 nm was measured. A standard curve was prepared from the absorbance of the sample of Zn-α2-glycoprotein, and the amount of Zn-α2-glycoprotein contained in each saliva sample was quantified from the standard curve.

  FIGS. 7-9 is the figure which showed the change of the density | concentration of Zn- (alpha) 2-glycoprotein in the saliva of the test subjects 5-7 before and after the Kraepelin work for every test subject, respectively.

  FIGS. 10-12 is the figure which showed the change of the density | concentration of the Zn- (alpha) 2-glycoprotein in the saliva of the test subjects 8-10 before and after ATMT for every test subject, respectively.

  From the above, it has been found that the concentration of Zn-α2-glycoprotein in saliva rises significantly after the Kraepelin work and ATMT, and the subject received by examining the concentration of Zn-α2-glycoprotein in saliva. It was suggested that the degree of stress could be determined with high sensitivity.

  The stress determination method of the present invention makes it possible to determine stress easily and objectively. Further, compared to a stress determination method using cortisol and chromogranin A, which are known stress determination markers, there is less variation between individuals, and the degree of stress can be determined with high sensitivity.

  In addition, the stress determination method of the present invention can use not only blood of the test animal but also body fluid collected non-invasively such as saliva, and the degree of stress received by the test animal can be easily and easily measured at home. Can be objectively determined.

Claims (8)

A method for determining the stress of a test animal,
A stress determination method for determining stress using the concentration of Zn-α2-glycoprotein in the body fluid of the subject animal as an index. An increase and a decrease in the concentration of the Zn-α2-glycoprotein are determined as an increase and a decrease in stress, respectively.
The stress determination method according to claim 1. A measurement step of measuring the concentration of Zn-α2-glycoprotein in the body fluid of the test animal;
A comparison step of comparing the concentration of Zn-α2-glycoprotein obtained in the measurement step with the concentration of Zn-α2-glycoprotein in the body fluid at rest;
A determination step of determining that stress has occurred when the concentration of Zn-α2-glycoprotein obtained in the measurement step is higher than the concentration of Zn-α2-glycoprotein in the body fluid at rest;
A stress determination method comprising:   The stress determination method according to claim 1, wherein the body fluid is a body fluid collected noninvasively.   The stress determination method according to claim 1, wherein the body fluid is saliva.   A marker for stress determination, comprising a Zn-α2-glycoprotein.   A diagnostic agent for stress determination, comprising an anti-Zn-α2-glycoprotein antibody. Receiving means for receiving information on the concentration of Zn-α2-glycoprotein in the bodily fluid of the test animal transmitted from the terminal;
Stress information storage means for storing information on the concentration of the Zn-α2-glycoprotein received by the receiving means;
Stress determination means for determining stress using as an index information on the concentration of the Zn-α2-glycoprotein stored by the stress information storage means;
Determination result transmission means for transmitting the determination result determined by the stress determination means to the terminal;
A stress judgment system.

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