JPH1123572A - Evaluating method for physiological state and simple physiological state evaluating kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evaluate the wide-range physiological state with high reliability by collecting a humor from a subject, and evaluating the physiological state correlative with the secretion quantity from the concentration of the chromogranins in the humor. SOLUTION: A humor as a measurement specimen is injected into a specimen loop 4, a valve 5 is switched, and the measurement specimen is sent to a reaction section 9 by a pump 8 via a carrier buffer solution 2. An antichromogranins primary antibody is fixed to the reaction section 9 in advance, and the chromogranins in the specimen is arrested. An enzyme-marked antichromogranins secondary antibody is injected into the specimen loop 4 and sent to the reaction section 9, both antibodies are reacted, the specimen not reacted and the antichromogranins secondary antibody are washed off, a valve 6 is switched, and an enzyme substrate solution 1 is sent out. The substrate reaction product by the marker enzyme arrested by the reaction section 9 is detected by a detector 10. The occurrence of the physiological state related with sympathetic nerve activity and tumor can be evaluated and diagnosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for evaluating a physiological condition and a kit for evaluating a simple physiological condition, and more particularly, is secreted into body fluids such as saliva, blood, urine, cerebrospinal fluid, and lymph fluid, and includes, for example, Various physiological conditions typified by physical stress or stress, prescribed medical diseases (eg, mental and nervous system diseases, or a wide range of diseases such as hypertension, endocrine disorders, various tumors, etc.) The present invention relates to a physiological condition evaluation method and a simple physiological condition evaluation kit for quantitatively evaluating the condition.

[0002]

2. Description of the Related Art Conventional methods for evaluating physiological conditions are described below.
Taking stress as an example, psychological evaluation in a questionnaire format, task evaluation methods such as flicker, electrophysiological measurement methods to measure brain waves and heart rate, and biochemistry to measure stress-responsive substances in blood and urine There are measurement methods and the like.

[0003] Each of the above methods has advantages and disadvantages. Among them, the biochemical measurement method has the advantage of being excellent in objectivity and quantitativeness because it measures the substance concentration. Catecholamines (adrenaline and noradrenaline) and cortisol, which are stress hormones, are often used as known substances to be measured in biochemical measurement methods.

[0004]

However, catecholamines and cortisols have hitherto been mainly used mainly for evaluation of stress, and are used for a wider range of various physiological conditions such as mental and nervous system diseases or hypertension. It is questionable whether it can be used for evaluating or diagnosing physiological conditions of a wide range of diseases such as endocrine disorders and various tumors.

[0005] Furthermore, as a substance to be measured only for stress evaluation, for example, adrenaline suitable for evaluation of mental stress and noradrenaline suitable for evaluation of physical stress have a sharper response and a shorter response time than cortisol. Has the advantage of being short, but has the drawback that it is difficult to measure in saliva or the like due to the problem of insufficient sensitivity, so that only means of highly invasive blood sampling is available. Conversely, cortisol can be measured non-invasively in saliva, but has a problem with responsiveness.

Accordingly, the present invention can evaluate a wide range of various physiological conditions, including evaluation of stress or load state, and can optionally employ non-invasive measurement using saliva, urine, etc. as a sample. It is an object of the present invention to provide a method for evaluating a physiological condition having no responsiveness problem such as cortisol, and to provide a practical device for performing the evaluation easily and quickly.

[0007]

[Point of view] The inventor of the present application has proposed that when a human is subjected to mental or physical stress or load, or that a human suffers from a wide range of diseases such as mental or nervous system diseases, hypertension, endocrine disorders and various tumors. When falling into a disease state, in a wide range of physiological conditions such as, saliva, blood, urine, cerebrospinal fluid, the concentration of chromogranin in body fluids such as lymph fluid, corresponding to the state that can be used as the original indicator substance, That is, it is found that it rises sharply in a state where quantitative evaluation is possible,
The present invention has been completed.

[0008]

[Means for Solving the Problems]

(Structure of the First Invention) A first invention of the present application for solving the above problems.
The structure of the invention (the invention according to claim 1) is to evaluate a physiological condition in which a body fluid is collected from a subject and a physiological condition having a correlation with the amount of secreted chromogranin is evaluated from an increase rate of chromogranin in the body fluid. Is the way.

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
A measurement piece in which an antibody fixing portion with a liquid storage structure is provided on the support and an anti-chromogranin antibody is fixed to the antibody fixing portion, and a substance for label expression prepared so as to be administrable to the antibody fixing portion, This is a simple physiological condition evaluation kit that is configured and used for evaluating a physiological condition.

[0010]

【The invention's effect】

(Effects of the First Invention) In the first invention, a wide variety of physiological conditions that could not be obtained can be evaluated by measuring the rate of increase of chromogranin in body fluids.
Then, since the concentration of chromogranin in the body fluid sharply increases in a state where quantitative evaluation is possible, a highly reliable evaluation result can be obtained.

Further, in the first invention, a body fluid such as saliva, blood, urine, cerebrospinal fluid, or lymph fluid can be arbitrarily selected or a body fluid of a type most suitable for the purpose can be selected as a specimen. And non-invasive measurements are easily possible,
Very useful.

(Effect of the Second Invention) According to the second invention, a practical device for easily and quickly evaluating the physiological condition according to the first invention is provided.

[0013]

Next, embodiments of the first invention and the second invention will be described.

(Physiological condition) The "physiological condition" to be evaluated in the present invention includes a wide range of various physiological conditions, both mental and physical. It includes various mental and physical physiological states that have not reached such a state. As the "physiological condition" in the present invention, at least at present, no positive ground to exclude a specific mental or physical physiological condition has been found.

One of the typical "physiological states" is mental or physical stress or stress,
These include, for example, mental stress, such as when nervous, physical stress due to exercise or physical work, etc.
There are stresses based on both mental and physical conditions, such as when driving a car for a long time, and when categorized in terms of load intensity and time, it cannot be called acute stress, chronic stress, or still stress Such a mental or physical load state is included.

In this regard, the following can be said.

It has been found that chromogranin in body fluids is co-secreted with catecholamines in blood, especially noradrenaline, and the secretion rate is correlated. Therefore, chromogranin in body fluids is an indicator of sympathetic nervous activity. From this, it is possible to evaluate and diagnose physiological conditions involving sympathetic nervous activity, medullary thyroid carcinoma, and the occurrence of tumors such as pheochromocytoma, using chromogranins in body fluids such as saliva, urine, and blood as indicator substances. it can.

Although not a function as an indicator of a physiological state, it has also been found that a chromogranin degradation product suppresses secretion of noradrenaline from chromaffin cells, and thus chromogranin also has a function of regulating stress. From this, it is conceivable to evaluate the physical or mental physiological state or the like by using the measurement of the chromogranin concentration in a body fluid, for example, saliva, urine, or blood.

Next, under a wide range of stresses such as mental stress such as tension stress and mental / physical stress such as driving stress related to driving of an automobile or the like, the chromogranin concentration in the body fluid may be reduced. It has been found that the stress increases quantitatively in response to the intensity of the stress. From this, it is possible to obtain, for example, evaluation data that is effective in terms of sensitivity engineering using chromogranin in a body fluid, for example, saliva, urine, or blood as an indicator substance. Examples of the use of such data include, for example, the design of office equipment and automobile interiors that do not stress the user, and the design of an acoustic or chromatic environment in a similar sense.

One of the other typical "physiological conditions" is a certain medical disease or condition.
The following are the targets. Nervous system disorders: Brain dysfunction, central nervous dysfunction, autonomic nervous dysfunction, etc. Mental illness: Mental disorders and the like. Endocrine disorders: Endocrine disorders, hyperparathyroid gland function or disorders. Various tumors: pheochromocytoma, pituitary tumor, neuroblastoma, endocrine organ tumor, etc. Cardiovascular diseases: hypertension, etc.

Regarding these, the following can be said.

In pheochromocytoma patients, about 8
With a statistically significant probability of ~ 90%, data have been obtained that show an abnormally high concentration of chromogranin in body fluids, corresponding quantitatively to the symptoms of cytomas, Chromogranin in urine, blood, cerebrospinal fluid and lymph can be a diagnostic indicator of pheochromocytoma.

[0023] Even in tumors of various endocrine organs, for example, small cell lung cancer, pituitary tumor, neuroblastoma, medullary thyroid carcinoma and the like, the symptoms of these tumor groups are quantified with a statistically significant probability. Correspondingly, data have been obtained that show high concentrations of chromogranin in body fluids,
For example, chromogranin in saliva, urine, blood, cerebrospinal fluid, and lymph serves as a diagnostic indicator for these tumor groups.

Even in patients with hypertension or hyperparathyroidism, data showing that chromogranin in body fluid shows a high concentration in a statistically significant probability in quantitative response to the symptoms of these tumor groups. Chromogranins in body fluids, such as saliva, urine, blood, cerebrospinal fluid, and lymph, are also indicators of the diagnosis of these conditions.

Data has been obtained that, even in patients with renal insufficiency, chromogranin in body fluids shows a high value with a statistically significant probability, and therefore chromogranin in body fluids, such as saliva, urine and blood, is diagnostic for these symptoms. It is also an indicator substance. Of the above-mentioned examples, the physiological state relating to mental or physical stress or stress state and the physiological state relating to certain medical diseases or symptoms are technically significant in applying the present invention. Are considered quite different.

The former is, for example, a design for office equipment or a vehicle interior that does not stress the user, or a sound or color in the same sense, for example, in terms of providing effective data in terms of perceptual engineering. This is because the latter provides effective data for medical treatment, while the use for the design of a medical environment and the like is considered promising.

Therefore, the following two inventions can be disclosed as inventions of the lower concept of the first invention.

[0028] 1-1: Invention: The constitution of the present invention is that a body fluid is collected from a subject, and the concentration of chromogranin in the body fluid is correlated with the amount of chromogranin secreted, which indicates a mental or physical stress or stress state. This is a method for evaluating a physiological condition for evaluating the condition. And the effect of this invention is
In addition to the effects of the first invention, mental or physical stress or stress can be evaluated,
The evaluation result can be effectively used for various purposes. As one example, use in so-called sensitivity engineering is conceivable. That is, for example, a design of office equipment or a car interior that does not give a stress to the user, a design of an acoustic or chromatic environment in a similar sense, and the like are conceivable.

[0029] 1-2th invention: The constitution of the present invention is to extract a body fluid from a subject and evaluate a predetermined medical disease correlated with the amount of chromogranin secreted from the concentration of chromogranin in the body fluid. This is a state evaluation method. And the effect of this invention is in addition to the effect of said 1st invention,
Mental and nervous system diseases, or the ability to evaluate the symptoms of a wide range of diseases such as hypertension, endocrine disorders, and various types of tumors. It is.

(Body Fluid) The "body fluid" to be collected in the present invention includes a wide variety of body fluids such as saliva, urine, blood, cerebrospinal fluid, and lymph. As the "body fluid" in the present invention, at least at present, no positive ground for eliminating a particular type of body fluid has been found.

When these various body fluids are viewed as being advantageous as so-called "specimens", the following can be generally said.

Saliva is the best in terms of ease of collection from a subject and non-invasiveness of a sample collection operation. Next, urine is a body fluid that is easy to use and blood is also easy to use when evaluating medical uses.

However, when matching between the type of physiological condition to be evaluated and the type of body fluid, that is, considering the quantitative correspondence between the two, there are cases where the cerebrospinal fluid, lymph and other body fluids are relatively superior. is there.

(Body Fluid Collection from Subject) In the first invention, the timing for collecting body fluid from the subject whose physiological condition is to be evaluated is particularly determined when the physiological condition to be evaluated is transient, such as temporary stress. Is an issue to consider.

However, generally speaking, the timing is not particularly limited. However, it is preferable that each subject of a plurality of experimental examples or comparative examples in the same evaluation test collect body fluid at the same appropriate timing.

If it is desired to conduct a more rigorous evaluation test, the body fluid is collected based on a curve of chromogranin secretion in body fluid (calibration curve) for the physiological state, which is known by a preliminary test of the evaluation test. May be determined.

Even when the physiological condition to be evaluated is a transient one such as temporary stress, for example, based on the same chromogranin secretion curve in the body fluid as above, the predetermined condition after the completion of the stress load is determined. It is preferable that the body fluid is collected after elapse of time.

As just one example of such a timing, immediately after the end of the transient stress load, or
Within 0 minutes is conceivable. At this timing, the amount of chromogranin secretion often reaches a peak or the amount of secretion reaches an amount that can be effectively evaluated. This also indicates the responsiveness of chromogranin.

(Chromogranin) Chromogranin A is known as chromogranin A and chromogranin B.
Secretogranin (chromogranin C) as a similar substance
There is a possibility that chromogranin substances that have not yet been elucidated may exist. However, the evaluation target of the present invention includes any of them.

The present inventor does not deny the possibility that decomposed products of these chromogranin substances can also be effective evaluation targets. However, in the case of an evaluation method using an anti-chromogranin antibody, the substance to be evaluated is limited to the range of substances that specifically bind to the antibody among the above various substances.

(Measurement of Chromogranin Concentration) As a method for measuring the chromogranin concentration in the body fluid, various known methods may be arbitrarily adopted.

One example is the antibody method. This method is a method in which chromogranin A, chromogranin B, chromogranin decomposing substance, and the like can be measured by using an anti-chromogranin A antibody, an anti-chromogranin B antibody, an anti-chromogranin decomposing substance antibody, and the like.

The antibody method is classified into an enzyme immunoassay using an enzyme, a radioimmunoassay using a radioactive substance, and a fluorescent antibody method using a fluorescent dye, depending on the method of labeling the antibody.

In addition, a direct method using an antibody of only one step (for example, separately preparing a predetermined amount of labeled chromogranin for competition and adding it to a body fluid sample to express the label, (Indirect method of knowing the chromogranin concentration in a body fluid sample) and an indirect method using a secondary antibody further labeled with respect to chromogranin captured by the antibody.

(Evaluation of Physiological Condition) The physiological condition can be evaluated based on the measurement results of the chromogranin concentration by comparing the experimental example of the present invention with an appropriate blank test.
The determination may be made based on the obtained chromogranin secretion curve data without performing the blank test. In the evaluation of stress and the like, psychological evaluation for the subject may be performed in parallel. For further accuracy, these methods may be used in combination.

(Simplified Physiological Condition Evaluation Kit) The support of the measuring piece in the simple physiological condition evaluation kit is not particularly limited in terms of its shape, dimensions, material, etc., but is made of, for example, a flat, slender and somewhat rigid material. It is preferred to use The liquid storage structure of the antibody fixing portion provided on the support may be any structure that does not cause the body fluid or the diluent administered to the antibody fixing portion to flow away. As an example, a cylindrical structure provided so as to surround the antibody immobilization part can be considered.

The antibody immobilization portion may be an anti-chromogranin antibody in the direct method or an anti-chromogranin primary antibody in the indirect method, which are immobilized by appropriate means, and the structure and surface shape are not particularly limited. For example, a planar chromogranin-fixing membrane provided on the support and surrounded by the liquid storage structure can be used.

The label expression substance prepared so as to be administrable to the antibody immobilization part together with the measurement piece constitutes a kit. Here, "prepared to be administrable" means that it is in a state where it can be distributed on the market as an integrated set with the measuring piece, taking into account the storage stability such as enclosing the container.

The “substance for expressing a label” includes an enzyme substrate, a fluorescent luminescent agent, and the like according to the classification of the antibody method described above.
The direct method includes, for example, a labeled competitive chromogranin, and the indirect method includes a labeled anti-chromogranin secondary antibody.

(High-speed measurement method) FIG. 1 shows a high-speed measurement method using flow injection analysis, which enables accurate quantification of chromogranin in about 10 to 20 minutes per sample.

In this method, first, a body fluid as a measurement sample is injected into the sample loop 4, the valve 5 is switched, and the measurement sample is supplied to the reaction section 9 by the pump 8 using the carrier buffer 2.
Send to Since the primary anti-chromogranin antibody is fixed to the reaction section 9, chromogranin in the sample is captured.

Then, an enzyme-labeled anti-chromogranin antibody (secondary antibody) is injected into the sample loop 4 and sent to the reaction section 9 in the same manner. After reacting both in the reaction section 9, the unreacted sample and the enzyme-labeled anti-chromogranin antibody are washed away, and the valve 6 is switched to send the enzyme substrate solution 1.

Thus, the measurement is performed by detecting the substrate reaction product of the labeling enzyme captured in the reaction section 9 by the detector 10. If the valve 7 is switched to send the regenerating solution 3 to remove the chromogranin or the enzyme-labeled anti-chromogranin antibody bound to the reaction section 9, the reaction section 9 can be regenerated for the next use.

[0054]

【Example】

(Method Used in Examples) The following Examples 1 and 2
In the measurement of chromogranin concentration in saliva,
At that time, the method of O'Connor et al. (Clin. Chem. 35, 1631-1
637, 1989). Specifically, the main points of the embodiment are as follows.

After collection, the cryopreserved saliva was naturally thawed at room temperature and centrifuged at 15,000 rpm for 10 minutes.
00 μl was used. Chromogranin labeled with ¹²⁵ I and anti-chromogranin antibody were added to each of them for 15 minutes.
After stirring, the mixture was stirred and left at 4 ° C. overnight. Then, 100 μl of rabbit serum was added thereto as a carrier antigen,
After stirring at room temperature for 5 minutes, 50 μl of a goat anti-rabbit γ globulin antibody solution was added, and the mixture was allowed to stand for 5 minutes after stirring.

Next, 1 ml of a polyethylene glycol solution was added thereto, and the mixture was allowed to stand for 10 minutes after stirring, and then centrifuged to precipitate an antigen-antibody complex. The supernatant was removed by suction, and the radioactivity of the precipitate was measured.

On the other hand, a calibration curve was prepared in the same manner using a chromogranin solution having a known concentration, and the chromogranin concentration in the sample was determined based on the calibration curve.

In Example 1, a questionnaire based on psychological evaluation of the subject was conducted in parallel with the measurement of the chromogranin concentration in saliva (feeling of tension). The score of the psychological evaluation in the questionnaire is 0-3 according to the report of the subject.
Is a relative evaluation of

In Example 2, the measurement of the concentration of cortisol in saliva was carried out in the same manner as in the case of chromogranin, and a questionnaire based on psychological evaluation of the subjects was also carried out (feeling of tension and fatigue). . The tension and fatigue scores of the psychological evaluation in the questionnaire were 0% before and after driving in the morning and afternoon.
"No feeling of tension or fatigue" is 0,
The evaluation was made with a score between 0 and 1, where "I feel strongly nervous or tired".

(Embodiment 1) This embodiment is an evaluation example of tension stress. That is, the change in salivary chromogranin concentration when a young researcher makes a presentation at an important meeting is shown in FIG. 2 together with a score for psychological evaluation.

As is clear from FIG. 2, it was confirmed that the salivary chromogranin concentration immediately after the presentation was more than twice as high as before the presentation. In a subsequent interview, the researcher replied, "I was very nervous about the presentation."

(Embodiment 2) This embodiment is an example of evaluating driving stress. That is, the salivary chromogranin concentration and the salivary cortisol concentration when a 37-year-old man traveled on the highway in the morning and afternoon for 2 hours each were measured, and a questionnaire based on psychological evaluation of the subjects was also conducted. The result is shown in FIG.

As can be seen from the plot of FIG. 3, the saliva sampling and psychological evaluation scoring were performed immediately before running in the morning and afternoon, one hour after starting running (middle point), and immediately after running.

From the data in FIG. 3, the chromogranin concentration in saliva is very sensitive to the load of driving stress and the score of psychological evaluation, and corresponds to no response time lag. The concentration of medium cortisol varies with a time lag of almost one hour.

From this, it can be seen that the chromogranin concentration in saliva is an excellent immediate stress indicator. On the other hand, it can be seen that the salivary cortisol concentration is an index that is more difficult to use because it requires accurate processing of the time lag in the evaluation in comparison with the chromogranin concentration.

(Embodiment 3) This embodiment shows an example of a measuring piece according to the simple physiological condition evaluation kit. 4 (a) and 4 (b), the measuring piece 11 is provided with a cylinder 13 constituting a liquid storage structure standing on one end side of a long and narrow rectangular plate-shaped support body 12 made of any rigid material. , A structure in which an antibody fixing portion 14 is provided at the inner bottom of the tube 13.

The antibody immobilization section 14 is a membrane, on which an anti-chromogranin antibody is immobilized. As evident in the figure,
A fixed volume reservoir is formed by the cylinder 13 above the antibody fixing portion 14. In addition, as described above, the measurement piece 11 constitutes a simple physiological condition evaluation kit in combination with a label expression substance (not shown).

The substance for expressing a label may be prepared as a simple ampule package made of, for example, glass or plastic, and the ampule package may be formed integrally with the measuring piece 11. Even if it is formed on the body, it is sufficient if it is enclosed in the same packaging box or the like as the measuring piece 11.

The contents of the label expression substance are also as described above. According to the measurement method, such as an enzyme substrate and a fluorescent agent, the direct method includes, for example, a labeled competitive chromogranin, the indirect method includes a labeled anti-chromogranin secondary antibody as necessary, and further, if necessary, saliva dilution Water for cleaning or washing may be included.

The method of using the simple physiological condition evaluation kit according to the present example is as follows, for example. That is, first, about 0.5 ml of the sample saliva and an equal amount of distilled water are added to the liquid reservoir on the antibody immobilizing section 14, lightly immersed, left standing for about 5 minutes, and then the liquid is discarded. . Then, about 0.5 ml of a solution of an enzyme-labeled anti-chromogranin secondary antibody is added, and the mixture is left standing for about 5 minutes. After washing the antibody-fixing portion 14 three times with 1 ml of distilled water, a coloring-type enzyme substrate solution is added and the solution is allowed to stand for about 5 minutes, and the physiological state is determined from the intensity of the coloring.

As can be seen from the above operation, the use of the simple physiological condition evaluation kit makes it possible to evaluate the physiological condition extremely easily without using any special equipment. Since the liquid storage structure is provided, there is no fear of spilling bodily fluids such as saliva to fail in measurement or soil the floor.

[Brief description of the drawings]

FIG. 1 is a diagram showing a flowchart of a high-speed measurement method for evaluating a physiological state.

FIG. 2 is a graph showing changes in salivary chromogranin concentration at the time of presentation of a study, together with scores for psychological evaluation.

FIG. 3 Chromogranin concentration in saliva when driving on a highway,
6 is a graph showing changes in salivary cortisol concentration and psychological evaluation score.

FIG. 4 is a view showing a simplified physiological condition evaluation kit according to the present invention, wherein FIG. 4 (a) is a plan view thereof, and FIG. 4 (b) is a sectional view taken along line AA of FIG. 4 (a). .

[Explanation of symbols]

 4 Sample loop 9 Reaction part 11 Measurement piece 12 Support 13 Tube 14 Antibody fixing part

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yukio Yamada 41-cho, Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central R & D Laboratories Co., Ltd. No. 41 at Yokomichi 1 Toyota Central Research Laboratory Co., Ltd. (72) Inventor Noboru Yanaihara 22 at 403 Kita, Shizuoka City, Shizuoka Prefecture

Claims (2)

[Claims] 1. A method for evaluating a physiological condition, comprising collecting a body fluid from a subject and evaluating a physiological condition having a correlation with the amount of chromogranin secreted from the concentration of chromogranin in the body fluid. 2. A measuring piece having an antibody fixing portion with a liquid storage structure provided on a support and an anti-chromogranin antibody fixed to the antibody fixing portion, and a label expression preparation prepared to be administrable to the antibody fixing portion. A simple physiological condition evaluation kit, comprising: a substance for evaluating physiological conditions.