JPH07159401A - Quantitative determination method by enzyme immunoreaction of cortisol in saliva - Google Patents

Abstract

PURPOSE:To achieve an accurate determination of cortisol in saliva with high sensitivity and high reproducibility without use of any radioactive isotope by mixing the saliva with an enzymejmarked cortisol and an anti-cortisol antibody to react. CONSTITUTION:Saliva is mixed with an enzyme-marked cortisol and an anti- cortisol antibody to react. Then, a mixture of a first reaction product of eortisol contained in the saliva and the anti-cortisol antibody contained in the saliva and a second reaction product of the enzyme-marked cortisol and the anti- cortisol antibody is separated from the anti-cortisol antibody left unreacted. Then, a color former and a coloring assistant are added to the mixture to measure absorbance. The enzyme used acts as a catalyst for enzyme reaction between the color former and the coloring assistant and includes peroxidase, alkali phosphatase and the like. The anti-cortisol antibody is produced in vivo by injecting cortisol (antigen) into blood of an immune animal such as rabbit, goat or the like and taken out in the form of serum.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying cortisol in saliva by an enzyme immunoreaction.

[0002]

2. Description of the Related Art Cortisol is a major glucocorticoid hormone secreted by the adrenal cortex. About 90% of cortisol in the blood is corticosteroid binding globulin (Corticos
teroid Binding Globulin), about 5% of which is bound to albumin, and the remaining about 5% is circulated in the body in the form of free (sugar metabolism, lipid metabolism, protein metabolism, water / electrolyte metabolism, It is deeply involved in the immune system. Therefore, when the cortisol concentration in blood is not normal, the metabolism and mechanism of these are abnormal. On the contrary, if cortisol in blood is quantified, abnormalities of these metabolisms and mechanisms such as hyperthyroidism or hypothyroidism (Cushing's syndrome), hypoadrenocorticism (Addison's disease), hypothalamus- It is possible to diagnose pituitary-adrenal cortex dysfunction and the like.

Further, there is a research report that the measurement of cortisol concentration is useful for assisting diagnosis of "depression" and sleep disorder syndrome and evaluation of its treatment (Hanada, K., Yamada,
N., Shimoda, K., Takahashi, K., Takahashi, S .: Direct
radioimmunoassay of cortisol in salivaand its appli
cation to the dexa methasone suppression test in af
fectivedisorders.Psychoneuroendocrinology, 10: 193-
201, 1985.).

There is also a research report that the measurement of cortisol concentration is useful for elucidating the neuropsychiatric mechanism in the brain including circadian rhythm (Spanger, G., Theeme.
rgence of adrenocortical circadian function in new
born and infantsand its relationship to sleep, fee
ding and maternal adrenocortical acti-vity. Early
Hun. Dev., 25: 197-208, 1991).

In order to quantify cortisol in blood, it is necessary to collect blood each time. In addition, in order to use cortisol concentration for diagnosis and evaluation, it is necessary to quantify at least every 4 hours in the case of diagnosis of diurnal rhythm abnormality. This is a great pain for the patient. Therefore, a method for quantifying cortisol in saliva has been proposed. A high correlation between cortisol levels in blood and salivary levels (cortisol in saliva is said to be 5% to 10% of that in blood (serum)) has already been demonstrated. (Umeda, T., Hiramatsu, R., Iwaoka, T.,
Shimada, T., Miura, F., Sato, T .: Use of saliva for
monitoring unfound free cortisollevelsin serum. Cl
in. Chim. Acta., 110: 245-253, 1981). There are other advantages to quantifying salivary cortisol. <Clinical advantages> (a) Unlike cortisol in serum which is affected by transcortin (cortisol-binding globulin), which is changed by pregnancy and administration of estrogen, saliva in which circulating protein-free cortisol is secreted Cortisol in the blood is "bioactive free (fre
e) Cortisol ”. (B) Psychological stress when collecting blood with an injection needle (stress)
Stimulation stimulates the secretion of cortisol, avoiding the problem that normal conditions cannot be quantified. (C) Informed concent of painful blood sampling
Even for psychiatric patients and children who have difficulty in obtaining cortisol, it is easy to obtain consent, and cortisol can be quantified. <Practical Advantages> (d) Since blood collection is unnecessary, it is easy to collect a sample (saliva). (E) The sample (saliva) can be collected at home by the patient (subject). (F) It is suitable for infants and children because it does not require painful blood sampling. (G) Since there is no painful blood sampling, it is suitable for diurnal variation tests that require frequent sample collection. (H) Unlike cortisol in serum, cortisol in saliva is not bound to a protein, so that saliva can be directly used as a sample. In other words, it is not necessary to perform pretreatment for protein removal with glutamic acid or the like before quantification.

Conventionally, the quantification of cortisol in saliva has been carried out exclusively by the RIA (radioimmunoassay) method. For example, Yamada, N., Jpn.J.Psychopharmaac
ol.7: 295-306 (1987). The RIA method uses cortisol labeled with a radioactive isotope, such as ¹²⁵ I (hereinafter abbreviated as ¹²⁵ I-labeled cortisol). The quantification method by the RIA method is the first step: mixing saliva, ¹²⁵ I-labeled cortisol and anti-cortisol antibody and reacting them; second step: the first step of cortisol and anti-cortisol antibody contained in saliva Centrifuging the reaction product and the mixture of the ¹²⁵ I-labeled cortisol and the second reaction product of the anti-cortisol antibody from the unreacted anti-cortisol antibody; (the unreacted anti-cortisol antibody becomes the supernatant, and the mixture is Since it becomes a precipitate, the supernatant is discarded. This separation step separates the reaction or bound constituting the mixture from the unreacted or free anti-cortisol antibody. This is referred to as BF separation) Third step: measuring the radioactivity of the mixture with a γ-counter.

[0007] Cortisol of various known concentrations was prepared in parallel, and a calibration curve (standard curve) showing the relationship between cortisol concentration and radioactivity was prepared, and the radioactivity measured in the third step was used in the saliva sample. The cortisol concentration of can be calculated.

[0008]

However, the RIA
The method for quantifying cortisol by the method has the following problems. (1) Because radiation is emitted, it can be quantified only at specific facilities based on the law. (2) Maintenance costs for the above facilities are high. (3) Since it deals with radiation, only those who have the required qualifications under the law can quantify. (3) Since radiation is emitted, equipment, gloves,
When discarding containers, solutions containing radioisotopes, etc., it is necessary to strictly dispose of them in accordance with the law, which increases treatment costs.

[0009]

Accordingly, the present invention provides a method for quantifying cortisol in saliva by an enzyme immunoreaction, which comprises the following first to fourth steps. First step: mixing saliva, enzyme-labeled cortisol and anti-cortisol antibody and reacting; second step: first reaction product of cortisol contained in the saliva and the anti-cortisol antibody, and the enzyme-labeled cortisol A step of separating a mixture of the second reaction product of the above-mentioned anti-cortisol antibody with unreacted anti-cortisol antibody; a third step: a step of adding a color former and a color assistant to the mixture; a fourth step: absorbance Measuring step;

[0010]

In the first step of the present invention, saliva, enzyme-labeled cortisol and anti-cortisol antibody are mixed. In this case, mix the three in this order or simultaneously. The enzyme-labeled cortisol is a product obtained by binding (combining) an enzyme and cortisol, and is available as a commercial product. The enzyme functions as a catalyst for enzymatically reacting a color-forming agent and a color-forming auxiliary agent described below, and examples thereof include peroxidase and alkaline phosphatase. The type of reaction differs depending on the enzyme, peroxidase catalyzes the oxidation reaction, and alkaline phosphatase catalyzes the hydrolysis reaction.

The enzyme-labeled cortisol is a 2- to 5-fold diluted enzyme-labeled cortisol (referred to as the "reference solution" in Example 1) for quantifying serum cortisol, that is, a concentration of 2.5 to 7 mg / It is preferable to use a solution diluted to the ml position because the reproducibility is good and the calibration curve (standard curve) is stable. Enzyme-labeled cortisol is 50 μl of saliva
About 0.1 to 0.5 mg (microliter), especially 0.13
It is preferable to mix about 0.32 mg. On the contrary, if at most, at least too much, there is a risk of not competing with “cortisol in saliva”.

[0012] When cortisol (antigen) is injected into the blood of immunized animals such as rabbits, goats, guinea pigs (guinea pigs), cattle, and sheep, anti-cortisol antibodies are produced in the animal's body in order to resist it. It is obtained by taking it out in the form of serum. Sometimes called simply cortisol antiserum. Since it is difficult to produce antibodies with cortisol alone, BSA (bovine serum albumin Bov
Injected (immunized) rabbits with cortisol combined with ine serum albumin) and extracted anti-cortisol BSA rabbit serum and the like are commercially available.

The anti-cortisol antibody has a concentration of 10 to 16 mg / ml.
It is easy to use the diluted product. The anti-cortisol antibody is mixed more than “the expected sum of cortisol in saliva and enzyme-labeled cortisol”.
It is preferable to mix anticortisol antibody in an amount of 0.5 to 1.0 mg, particularly 0.64 to 0.69 mg, with respect to 50 μl (microliter) of saliva.

The mixing is carried out by using cortisol contained in saliva as an antigen to cause an antigen-antibody reaction with the anti-cortisol antibody (herein referred to as the first reaction), and using enzyme-labeled cortisol as an antigen, the anti-cortisol antibody and the antigen-antibody reaction. (Here, it is referred to as the second reaction). The two reactions are competing reactions. Therefore, in order to fully carry out the reaction, after mixing, the reaction time (incubation)
It is preferable to incubate for 3 to 24 hours. Particularly, when about 0.13 mg of enzyme-labeled cortisol is mixed with 50 μl of saliva, it is preferable to leave it for 16 to 24 hours, and when about 0.32 mg of enzyme-labeled cortisol is mixed with 50 μl of saliva, 3 to 3 It is preferable to leave it for 5 hours.

After the reaction, a reaction product of cortisol (indicated by ●) contained in saliva and an anti-cortisol antibody (indicated by Y) (herein referred to as first reaction product (● Y))
And a reaction product of the enzyme-labeled cortisol (indicated by ● ^* ) and the anti-cortisol antibody (Y) (herein referred to as a second reaction product (● ^* Y)) is produced. The first reaction and the second reaction are competing reactions, and the first reaction product (● Y) and the second reaction product are mixed with the first reaction product (● Y) at a ratio equal to “the ratio of the amount of cortisol in saliva to the enzyme-labeled cortisol” present in the field. Reaction product (●
^* Y) is generated.

In the second step, the mixture of the first reaction product (Y) and the second reaction product ( ^* Y) is separated from unreacted or free anti-cortisol antibody. There are several separation methods. A convenient method is to use a second immobilized
It uses an antibody. The second antibody is produced in the body of an animal to resist it when the anti-cortisol antibody is used as an antigen and injected into the blood of another immunized animal. For example, when the anti-cortisol antibody is made from rabbit (rabbit IgG), "anti-rabbit IgG goat antibody" produced from goat is used as the second antibody. A plate on which the immobilized second antibody is immobilized is commercially available. The commercially available product is called "Enza Plate Cortisol" and measures 8 cm in length x 12 cm in width x 13 mm in thickness.
6m diameter on the plastic plate called "well"
Twelve m-shaped depressions are provided at regular intervals in the lateral direction,
Eight horizontal rows are provided at regular intervals in the vertical direction. The well volume is 300 μl and the total number of wells is 48. Inner surface of each well (up to 200 μl volume level)
The “immobilized second antibody” is immobilized on.

Add the mixing or reaction products that have completed or are in the first step to this well, or
Saliva, enzyme-labeled cortisol and anti-cortisol antibody may be directly poured into the wells to mix and react. In the latter case, the first step and the second step are not distinguished in terms of processing or operation, but this is also within the scope of the present invention (as defined in claim 10).

In the former case, the first reaction product (Y) and the second reaction product ( ^* Y) in the well, but this time, the respective reaction products (Y) and ( ^* Y) the cause second antibody (Y ₂₎ and the antigen-antibody reaction as an antigen. Also in this case, the respective reactions compete with each other and the two reaction products (● Y
Y ₂ ), (● ^* YY ₂ ). Two products (●
The amount ratio of (YY ₂ ), (● ^* YY ₂ ) is the first in the well.
It is equal to the amount ratio of the reaction product (● Y) and the second reaction product (● ^* Y). In the latter case, first in the well first
The reaction and the second reaction occur in competition with each other, and thereafter, the respective reaction products occur in competition with the second antibody. Also in this case, the quantitative ratio of the two kinds of products (● YY ₂ ) and (● ^* YY ₂ ) generated by the reaction with the second antibody is determined by the first reaction product (● Y) and the second reaction product. It becomes equal to the quantity ratio of the object (● ^* Y).

Since the two kinds of products produced by the reaction with the second antibody are immobilized on the inner surface of the well, the contents in the well are discarded by inverting the plate. The discarded contents include unreacted or free anti-cortisol antibody, first reaction product, second reaction product, second antibody and the like. The second step is also a type of BF separation, but in addition to the second antibody method using the second antibody (in addition, for example, there is also a γ-globulin precipitation method), the elution method (for example, ammonium sulfate method, polyethylene glycol method). Method), adsorption method (for example, DCC method), protein precipitation method (for example, method using BSA or gelatin) and the like.

The third step uses all or part of the separated "mixture of _two products (● YY ₂ ), (● ^* YY ₂ )". Here, a color former and a color assistant are added to this mixture. In the method using the immobilized second antibody, a color former and a color assistant may be poured into the well. Color developing agent is the catalytic action of enzyme-labeled cortisol (enzymatic reaction)
Depending on the color developing aid, it develops color. It is also called an enzyme substrate because it is entrusted to the enzymatic reaction. As used herein, the term "color formation" means that the product produced by the reaction of the color former is
Absorption of specific light from the infrared region to the visible light region to the ultraviolet region or emission of fluorescence. Color formers include, for example, O-phenylenediamine, P-nitrophenyl phosphate (phosphoric acid monoester of P-nitrophenol), and the like. The former reacts with hydrogen peroxide (color-forming aid) by peroxidase as an enzyme to be oxidized and develop color. The latter is hydrolyzed with water (coloring auxiliary) by alkaline phosphatase as an enzyme to produce P-nitrophenol (yellow with absorption at 400 to 420 nm).

As described above, the color-forming aid reacts with the color-forming agent by an enzymatic reaction to cause the color-forming agent to develop a color.
The color former, the color assistant and the enzyme have a special relationship, and the reaction between the color former and the color assistant is quantitatively catalyzed by the amount of enzyme.
There is a correlation between the amount of enzyme and the colored reaction product. In order to sufficiently react the color forming agent and the color forming auxiliary agent, it is preferable to leave them for a reaction time of about 30 to 1 hour.

The reaction between the color-forming agent and the color-forming assistant is described as follows: "The reaction product of the second reaction product (● ^* Y) and the second antibody (Y ₂ ) (●
^* YY ₂ ) ”has an enzyme (the enzyme is derived from the enzyme-labeled cortisol ● ^* ) and is quantitatively caused. Therefore, the amount of the colored product is proportional to “the reaction product of the second reaction product and the second antibody (● ^* YY ₂ )”. Conversely, the amount of colored product is inversely proportional to the amount of cortisol in saliva (specimen).

Since the cortisol concentration in saliva can be predicted to be within a predetermined range by the conventional method, the maximum amount should be considered.
Further, the amount of enzyme-labeled cortisol used, the second used
The stoichiometric amounts of the required color-forming agent and color-forming auxiliary can be calculated by considering the amount of antibody and the like. The amount of the colored oxidation product can be easily measured with an absorptiometer, a colorimeter, a spectrophotometer, a fluorescence analyzer and the like.

Absorbance (or color development) thus measured
To a calibration curve (standard curve) prepared in parallel using various known concentrations of cortisol,
The cortisol concentration (μg / dl) of saliva (sample) is calculated. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0025]

Example 1 (1) Reagents or reagents used: a) Standard cortisol (STD): Cortisol stock solution (10.05) sold by Ciba Corning Diagnostics (hereinafter abbreviated as CCD) for serum test. mg
/ 100 ml ethanol solution) was prepared. Hereinafter, the undiluted solution of cortisol refers to this.

Next, the stock solution of cortisol was added to the buffer solution.
Various standard cortisol (STD) having the following concentrations were prepared by diluting with. The buffer solution is phosphate buffered saline containing 0.1% bovine serum albumin (pH 7.2 to 7.4; Ph.
osphate-Buffered Saline0.1% Bovine Serum Albumin
: PBS-BSA) was used. Hereinafter, the buffer solution refers to this.

0.00, 0.04, 0.08, 0.16, 0.31, 0.63, 1.
25, 2.50 (unit: μg / dl) b) Enzyme-labeled cortisol standard solution: “Enzyme-labeled cortisol (white lyophilized product, enzyme is peroxidase; purity; 90% or more; enzyme amount / cortisol ≈ 0.5) ”in 1 vial (1
I prepared it. One vial contains about 80 mg of enzyme-labeled cortisol, which is dissolved in 6 ml of buffer solution,
An enzyme-labeled cortisol standard solution (about 13 mg / ml) was prepared. It is stable for 1 week at 2-8 ° C.

C) Enzyme-labeled Cortisol Diluted Solution Enzyme-labeled Cortisol Diluted Solution 2 (about 6.5 mg / about 2 mg) prepared by diluting the enzyme-labeled Cortisol standard solution prepared in the previous section twice with a buffer solution (dilution rate: × 2). ml) and an enzyme-labeled cortisol diluted solution 5 (about 2.5 mg / ml) diluted to 5 times (dilution rate: × 5) were prepared.

D) Anti-cortisol antibody: 1 vial of "anti-cortisol BSA rabbit serum (white lyophilized product)" sold by CCD Co. for serum test was prepared.
The vial contained about 80 mg of anti-cortisol antibody, which was dissolved in 6 ml of buffer solution to prepare a solution (about 13 mg / ml). It is stable for 1 week at 2-8 ° C.

E) Second antibody: "Enzaplate Cortisol (well number: 48)" sold by CCD Co. for serodiagnosis was used. Hereinafter, this is simply called a plate. The plate contains "anti-rabbit IgG" as the second antibody.
“Goat antibody” is immobilized on the surface of each well. The amount of secondary antibody in each well is 10 μg.

F) Coloring agent: 16 mg of O-phenylenediamine (white lyophilized product) was weighed, and this was used as a coloring aid 16 below.
It was dissolved in ml (concentration: 1 mg / ml) and used. Since the color former is vulnerable to oxygen and light, it was dissolved so as not to generate bubbles, and it was stored in a cool and dark place and used within 30 minutes. g) Color development aid: 0.1 M phosphate-citrate buffer containing 0.015% hydrogen peroxide was used.

H) Washing solution: Take 100 ml of a concentrated washing solution (phosphate buffered sodium chloride containing a small amount of surfactant) sold by CCD Company, and add purified water to this to make 1000 ml to prepare a washing solution. did. (2) Preparation of calibration curve: Eight kinds of standard cortisol (STD) prepared in the previous item a) were divided into two groups for the double examination, and the first group (OD492-1) and the second group (OD492- 1)

After removing the solution in the wells of the plate, the solution is completely removed by inverting and tapping on blotter paper. Then, 50 μl of the standard cortisol (1st group, 2nd group) from the previous section was dispensed into each well, and then 50 μl of enzyme-labeled cortisol diluent 2 or diluent 5 was added to each well.
Dispense 50 μl each of anti-cortisol antibody. Shake the plate gently and mix the contents of the wells.

Incubate at 25 ° C. for a predetermined time as an incubation time. After the contents of each well are removed by inverting the plate, the wells are washed 3 times with the washing solution. Dispense 150 μl of color developer into each well. Incubate for 30 minutes at 25 ℃ in the dark.

In order to stop the reaction, 150 μl of 1N sulfuric acid aqueous solution was added to each well and mixed gently with hot water. After stopping the reaction, the plate is inverted to remove the content liquid in each well, and then the plate is set as it is in a colorimeter "Bering ELISA Processor" manufactured by Hoechst, and the absorbance is measured at a wavelength of 492 nm.

The average absorbance (AVG OD) of standard cortisol (STD) having a cortisol concentration of 0 μg / dl was A ₀ ,
The absorbance of wells which only standard cortisol (STD) did not dispense a blank A _B, cortisol concentration
Using the average absorbance (AVG OD) of standard cortisol (STD) of 0.04 μg / dl or more as A _x , the relative binding rate (B / B ₀ ) is determined by the following formula.

B / B ₀ = [(A _x −A _B ) / (A ₀ −A _B )] × 100 (%) For various standard cortisol (STD) whose absorbance was measured, the relative binding rate is represented by the ordinate (Y Axis), cortisol concentration (μg / dl) is plotted on the coordinate with the horizontal axis (X axis),
A calibration curve (actually a computer-calculated equation) is created by logarithmically converting the cortisol concentration. (3) Quantification of cortisol: standard cortisol (ST
Using a sample instead of D), perform the same operation as in the previous section (2), measure the absorbance of the first and second groups for that sample, calculate the average absorbance (AVGOD), and perform parallel analysis under the same conditions. The cortisol concentration of the sample is calculated (quantified) from the prepared calibration curve. (4) Examination of optimum conditions: In order to determine the optimum conditions of the amount of enzyme-labeled cortisol and the reaction time (incubation time), as a sample, a) standard cortisol (ST) in the previous section (1) was used.
Various cortisol diluted solutions prepared in the same manner as in D) were prepared. Since these specimens have known cortisol concentrations, they are hereinafter referred to as known-concentration specimens.

With respect to specimens of known concentration, the concentration of enzyme-labeled cortisol and the reaction time (incubation time) were changed, and the operation of the above (3) was repeated to quantify the cortisol concentration for each. Some of the results are reported below. a) As enzyme-labeled cortisol, the enzyme-labeled cortisol diluent 2 (about 6.5 mg /
ml) and the reaction time (incubation time) was set to 3 hours (Table 1): The cortisol concentrations of known samples are shown in the column labeled "STD (μg / dl)" in Table 1 below.

The absorbance of a sample of known concentration was measured according to the preceding paragraph (3), the measured absorbance of the first group is shown in the column labeled "OD492-1" in Table 1, and the absorbance of the second group is shown in Table 1. Is shown in the column labeled "OD492-2", and the average value of these absorbances (average absorbance) is shown in Table 1 in the column labeled "AVG OD". For samples of known concentration, the cortisol concentration of the sample was calculated from the calibration curve. The calculated values up to the second decimal point are shown in the column labeled "CALCULATED (μg / dl)" in Table 1.

The difference rate between the calculated value and the cortisol concentration of the sample of known concentration (that is, the cortisol concentration in the column labeled "STD (μg / dl)" in Table 1) is shown in Table 1 "DIFFERENCE".
(%) ”Is shown in the column labeled. Table 1 shows “Averege difference
(%) ”Is shown in the column labeled. The maximum value of the difference rate is also shown in the column labeled "Range max (%)" in Table 1, and the minimum value is shown in the column labeled "Range min (%)" in Table 1.

[0041]

[Table 1]

B) As the enzyme-labeled cortisol, the enzyme-labeled cortisol diluting solution 2 with a dilution ratio of 2 from the standard solution
(About 6.5 mg / ml) and the reaction time was 6 hours (Table 2): The same measurement as in a) was repeated except that the reaction time was changed to 6 hours. The results are shown in Table 2 below.

[0043]

[Table 2]

C) As enzyme-labeled cortisol, an enzyme-labeled cortisol diluting solution 2 having a dilution ratio of 2 from the standard solution
(About 6.5 mg / ml) and the reaction time was 24 hours (Table 3): The same measurement as in a) was repeated except that the reaction time was changed to 24 hours. The results are shown in Table 3 below.

[0045]

[Table 3]

D) As the enzyme-labeled cortisol, the enzyme-labeled cortisol diluent 5 with a dilution ratio of 5 from the standard solution
(About 2.6 mg / ml) and the reaction time was 3 hours (Table 4): The same measurement as in a) was repeated except that the enzyme-labeled cortisol diluent 2 was replaced with the diluent 5. The results are shown in Table 4 below.

[0047]

[Table 4]

E) As the enzyme-labeled cortisol, the enzyme-labeled cortisol diluent 5 with a dilution ratio of 5 from the standard solution
(About 2.6 mg / ml) and the reaction time was 6 hours (Table 5): The same measurement as in d) was repeated except that the reaction time was changed to 6 hours. The results are shown in Table 5 below.

[0049]

[Table 5]

F) As the enzyme-labeled cortisol, the enzyme-labeled cortisol diluent 5 with a dilution ratio of 5 from the standard solution
(About 2.6 mg / ml) and the reaction time was 24 hours (Table 6): The same measurement as in d) was repeated except that the reaction time was changed to 24 hours. The results are shown in Table 6 below.

[0051]

[Table 6]

A condition in which the average value of the difference rates (Averege difference) is small can be said to be a preferable quantitative condition. As can be understood from these results (Tables 1 to 6), the conditions for the amount of enzyme-labeled cortisol and the reaction time were as follows: the former: saliva 50μ
About 0.13 mg (= about 2.6 mg / ml × 50 μl) for l, the latter: 16 to 24 hours, and the former: about 50 μl for saliva, about 0.
32 mg (= about 6.5 mg / ml × 50 μl), latter: 3 to 5 hours is preferable.

If the amount of enzyme-labeled cortisol is too high,
There is a possibility that the competitive reaction with cortisol to be quantified is inhibited and the quantification is not performed correctly.

[0054]

[Example 2] (1) Collection of saliva (specimen): Targeting 19 preterm premature infants and 16 term infants, each aged 2 years and 6 months, from 16:00 to 4 hours at 24 hours or 28 hours in some cases 100 μl or more of natural saliva was collected at home from one subject at a total of 7 times or 8 times in total. The collection time was within 1 hour before and after that.

The subject baby gave saliva directly to the Eppendorf tube, or if it could not do so, the mother used a plastic dropper to collect the natural saliva stored in the mouth floor of the subject baby. Immediately after saliva collection, saliva frozen in a home refrigerator was taken within 24 hours and stored frozen at -20 ° C. When the amount of saliva was small, a small amount of ascorbic acid was put into the oral cavity of the target baby to stimulate saliva secretion and then collected.
Since the protein stored in the salivary glands is secreted, its concentration changes depending on the secretory amount and the secretory rate.
However, as for cortisol in saliva, only free cortisol, which has no binding protein in circulating blood, is secreted by passing through salivary glands, and therefore cortisol circulating in blood is secreted. The concentration is considered to be unchanged regardless of the changes in the secretion amount and the secretion rate due to stimulation.

In order to confirm that the cortisol concentration in saliva does not change depending on the presence or absence of irritation, adult 51
For each subject (30 males and 21 females), 100 μl or more of saliva was collected before and after stimulation with ascorbic acid. Further, in order to confirm whether or not the cortisol concentration in saliva measured by the quantitative method of the present invention reflects the cortisol concentration in blood (serum), cortisol in saliva is confirmed.
The correlation between serum concentration and serum cortisol concentration was investigated.
100 μl of saliva per person for 4 subjects (2 males and 2 females) of adults A to D at the same time as the target children.
After the above collection, 1 ml of blood was collected immediately thereafter. After collection, it was subjected to 3000 rpm centrifugation for 5 minutes, and the supernatant (serum)
Was stored frozen at -20 ° C. (2) Reagents or reagents used: a) Standard cortisol: This is necessary for preparing a calibration curve. When quantifying cortisol in saliva (specimen), always measure the absorbance of cortisol at a known concentration in parallel. Need to measure. This is because the conditions and the calibration curve change each time.

Various standard cortisol having the following concentrations were prepared by diluting a stock solution of cortisol (10.05 mg / 100 ml ethanol solution) with a buffer solution. [For saliva (unit: μg / dl)] 0, 0.039, 0.078, 0.
156, 0.312, 0.625, 1.25, 2.50 8 types [for serum (unit: μg / dl)] 0, 1.6, 3.1, 6.3,
12.5, 25, 50 7 types b) Enzyme-labeled cortisol: The enzyme-labeled cortisol diluted solution 5 (2.6 mg / ml) prepared in Example 1 was prepared.

C) Others: The anti-cortisol antibody (anti-cortisol BSA rabbit serum) used in Example 1 and the second
An antibody (plate), a coloring agent (including a coloring aid), and a washing solution were prepared. (3) Quantification of cortisol in saliva: The saliva that had been frozen and stored was thawed at room temperature, then centrifuged at 3000 rpm for 5 minutes, and the supernatant was directly used for measurement as a sample. No pretreatment was done.

After removing the solution in the wells of the plate, the solution was completely removed by inverting and tapping on blotter paper. 1 set (Because of double inspection, 1 set is 2 pieces below)
Put 50 μl of the saliva (specimen) from the previous section into the wells of the above section, and use another set of 8 wells with different concentrations to prepare the calibration curve (2).
50 μl of each standard cortisol (known concentration) prepared in step a) was added to each well, and then 50 μl of enzyme-labeled cortisol diluent 5 and 50 μl of anti-cortisol antibody were added to each well.
μl was dispensed separately.

As a blank, one set of wells (without standard cortisol) was diluted with enzyme-labeled cortisol 5 solution.
50 μl and 50 μl of anti-cortisol antibody were dispensed respectively. The plate was shaken gently and the well contents were mixed. Incubate for 16-24 hours at 25 ° C. The contents of each well were removed by inverting the plate, and then the wells were washed 3 times with a washing solution.

150 μl of the color former was dispensed into each well. It was allowed to incubate at 25 ° C for 30 minutes in the dark. In order to stop the reaction, 150 μl of 1N sulfuric acid aqueous solution was poured into each well and mixed gently with hot water.

After the reaction was stopped, the plate was inverted to remove the content liquid in each well, and then the plate was set as it was in the above-mentioned colorimeter "Bering ELISA Processor", and the absorbance was measured at a wavelength of 492 nm. From the measured absorbance, the relative binding rate (B / B ₀ ) = [(A _x −A _B ) / (A ₀ −A)
_B )] × 100 was determined, a calibration curve was prepared from standard cortisol (known concentration), and the cortisol concentration in saliva (sample) was calculated based on the calibration curve. (4) Quantification of cortisol in serum: The frozen serum was thawed at room temperature, and then 10 μl of serum and 10 μl of a) in (2) above were pretreated to separate cortisol and protein. Add 200 μl of each aqueous glutamic acid solution to each standard cortisol for serum (known concentration) prepared in step 1, and mix at room temperature.
The samples were left for a minute (incubate) and used as samples.

The serum (specimen) of the previous section was added to one set of wells.
50 μl of the standard cortisol for serum (known concentration) prepared in (a) of (2) above was added to each of 7 wells, and 50 μl of the enzyme-labeled cortisol standard solution 5 was added to each well. 50 μl of anti-cortisol antibody
Each was dispensed. In addition, as a blank, 50 μl of enzyme-labeled cortisol standard solution and 50 μl of anti-cortisol antibody were dispensed into one set of wells (without standard cortisol).

The plate was gently shaken to mix the contents of the wells. It was incubated at 25 ° C. for 4 hours. The rest is the same as for saliva. Finally, a calibration curve was created from the absorbance of standard cortisol (known concentration), and the cortisol concentration in serum (sample) was calculated based on the calibration curve. (5) Results a) Cortisol concentration in saliva: 35 × 7 = 245 collected from a total of 35 target infants, 19 preterm premature infants and 16 term infants
Of the samples, a sufficient amount of saliva (sample) was obtained 2
There were 42 specimens. The cortisol concentrations of these 242 samples are shown in Tables 7 to 10 below.

[0065]

[Table 7]

[0066]

[Table 8]

[0067]

[Table 9]

[0068]

[Table 10]

As is clear from the above results, the cortisol concentration in saliva of the target infant was 0.001 to 2.252 μg / dl.
90% of which was in the range of 0.03 to 0.78 μg / dl. b) Diurnal variation of cortisol concentration in saliva: The measured values in Tables 7 to 10 are summarized in FIG. As shown in Fig. 1, there was a significant diurnal variation with time in each group of premature premature infants and term infants (premature premature infants one-way F = 5.43, P = 0.000).
1; term infant F = 5.12, P = 0.0001), and as shown in Tables 9 to 10, circadian rhythm was also observed in the premature premature infant group.

As can be understood from these results, there was a significant difference in the amplitude of diurnal variation, that is, the highest (top) and the lowest (bottom) cortisol (premature premature infant t = 6). .
18, P = 0.0001; term infants t = 4.41, p = 0.0006). The amplitude of diurnal variation for each individual was 36% to 105% for CV (mean CV 72 ± 22%). When expressed as the mean value of “cortisol concentration in saliva” every 4 hours in each group of premature preterm infants and term infants, both groups showed the lowest value at midnight and the highest value at 8 am. It was There was no significant difference in the diurnal variation pattern between the two groups (two-way ANOVA F
= 0.46, P = 0.84), indicating that premature premature infants have the same pattern of circadian rhythm as term infants (see Fig. 1).

C) Reproducibility during the same trial (intraassay varia
tion): Cortisol stock solution and DCC (Dextran Coated)
Char sal) treated saliva (hereinafter referred to as treated saliva) was prepared. Treated saliva is depleted of all steroids, including cortisol. By diluting the above cortisol stock solution almost twice with treated saliva, four types of specimens 1 to 4 were obtained.
Was prepared.

Each of these samples 1 to 4 was divided into 4 minutes, and cortisol was quantified in parallel on the same plate. The results are shown in Table 11 below.

[0073]

[Table 11]

According to this, for example, in the saliva of the specimen 1, the cortisol concentration was 1.781 μg / d in the first quantification.
l Second time 1.704μg / dl Third time 1.704μg / d
l The fourth time is almost equal to 1.667. The reproducibility between the same trials is the coefficient of variation 2.4% ~ 9.9% (mean mean = 5.8%, n =
4) was good. d) Interassay variation: Samples 1 to 7 having different concentrations were prepared by diluting cortisol stock solution almost twice with treated saliva. These samples 1 to 7 and the treated saliva itself (sample 8) were each divided into 10 minutes, and cortisol was quantified 10 times for the same sample by changing plates (plates 1 to 10) and times. The results are shown in the table below
Shown in 12.

[0075]

[Table 12]

According to this, for example, for sample 1, plat
The concentration of cortisol was 2.32 μg in the e (plate) 1 assay.
/ Dl, 2.32 μg / dl for plate 2, 2.06 μ for plate 3
It is almost equal to g / dl. The reproducibility between multiple trials has a coefficient of variation of 3.2% to 8.9% (mean mean = 6.6%, n = 10).
It was good. e) Minimum detection sensitivity: Samples 1 to 7 having different concentrations were prepared by diluting a stock solution of cortisol almost twice with treated saliva. Cortisol was quantified with respect to these samples 1 to 7 and the sample 8 (cortisol concentration 0) which is the treated saliva itself. This quantification was repeated 5 times in succession. And
The concentration at which the upper limit (average value + 2SD) of the measured values of sample 8 (0 concentration) and the lower limit (average value−2SD) of the measured values of samples 1-7 did not intersect was defined as the minimum detection sensitivity. As a result, the lowest detection sensitivity (sensitivity) was 4 pg / ml. f) Correlation between cortisol in saliva and cortisol in blood: Based on the above-mentioned measured values, graphs of daily fluctuation patterns of cortisol concentration in saliva and cortisol concentration in blood of 4 adults A to D Turned into The results are shown in FIG. 2 (adult A), FIG. 3 (adult B), FIG. 4 (adult C), and FIG. 5 (adult D). In the figure, ◆ indicates the cortisol concentration in saliva, and ● indicates the cortisol concentration in serum (Serum).

Then, the correlation between the cortisol concentration in saliva and the cortisol concentration in blood was subjected to regression analysis. As a result, as shown in FIG. 6, the correlation coefficient r = 0.857 and the regression equation y = 0.045.
A good correlation was confirmed with x-0.079, n = 27). In particular,
The fact that the regression coefficient was 0.45 means that "the cortisol concentration corresponding to 1/20 of the cortisol concentration in blood was measured from saliva by the quantification of this example". , Conforms to conventional dogma. Also, the intercept is 0.079 (≒
The fact that 0) is shown indicates that the quantification of this example does not measure other substances.

These results prove that the quantitative method of the present invention is accurate and can be sufficiently put to practical use.

[0079]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, cortisol in saliva can be accurately and reproducibly quantified without using a radioisotope. Therefore, various problems of the RIA method are solved, and quantification can be performed at low cost.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the time when saliva was collected and the concentration of cortisol in saliva.

FIG. 2 is a graph showing the relationship between the time when saliva and blood were collected for adult A and the cortisol concentration in saliva (Saliva) and serum (Serum).

FIG. 3 is a graph showing the relationship between the time when saliva and blood were collected from adult B and the cortisol concentration in saliva (Saliva) and serum (Serum).

FIG. 4 is a graph showing the relationship between the time when saliva and blood were collected for adult C and the cortisol concentration in saliva (Saliva) and serum (Serum).

FIG. 5 is a graph showing the relationship between the time when saliva and blood were collected from adult D and the cortisol concentration in saliva (Saliva) and serum (Serum).

FIG. 6 is a graph showing the correlation between the concentration of cortisol in saliva (Saliva) and the concentration of cortisol in serum (Serum). One scale on the horizontal axis is 5 μg / dl and one scale on the vertical axis is 0.2 μg / dl. that's all

Claims (10)

[Claims] 1. A first step: a step of mixing saliva, an enzyme-labeled cortisol and an anti-cortisol antibody and reacting them; a second step: a first reaction product of cortisol contained in the saliva and the anti-cortisol antibody; And a step of separating a mixture of a second reaction product of the enzyme-labeled cortisol and the anti-cortisol antibody from unreacted anti-cortisol antibody; a third step: a step of adding a color former and a color assistant to the mixture; And a fourth step: a step of measuring absorbance; a method for quantifying cortisol in saliva. 2. Saliva, enzyme-labeled cortisol and anti-cortisol antibody are poured into a well in which the immobilized second antibody is fixed, and the cortisol contained in the saliva is reacted with the anti-cortisol antibody. To produce a first reaction product, and at the same time to react the enzyme-labeled cortisol with the anti-cortisol antibody to produce a second reaction product, and then to produce the first reaction product and the second reaction product. Competing reaction with the second antibody (1); Discarding the contents in the well (2); Pouring a color former and a color assistant into the well (3); Contents in the well Method for quantifying cortisol in saliva, which comprises the step of discarding (4); and the step of measuring the absorbance of the well (5). 3. The saliva 50 containing the enzyme-labeled cortisol
3. The quantitative method according to claim 1, wherein 0.1 to 0.5 mg is mixed with μl. 4. The saliva 50 containing the enzyme-labeled cortisol
3. The quantification method according to claim 1, wherein 0.13 to 0.32 mg is mixed with μl. 5. The anti-cortisol antibody is added to the saliva 50 μm.
The quantitative method according to claim 1 or 2, wherein 0.5 to 1.0 mg is mixed with l. 6. The anti-cortisol antibody is added to the saliva 50 μm.
The quantitative method according to claim 1 or 2, wherein 0.64 to 0.69 mg is mixed with l. 7. The quantification method according to claim 1, wherein the reaction time in the first step or step (1) is 3 to 24 hours. 8. The reaction time in the first step is about 0.13 when the enzyme-labeled cortisol is added to 50 μl of the saliva.
It is 16 to 24 hours when it is mixed with mg, and it is 3 to 5 hours when about 0.32 mg of the enzyme-labeled cortisol is mixed with 50 μl of the saliva, which is 3 to 5 hours. Quantitative method described. 9. The quantification method according to claim 1, wherein the second step is a step of separating using a solid-phased second antibody. 10. The quantitative determination according to claim 1 or 2, wherein the enzyme of the enzyme-labeled cortisol is peroxidase, the color former is O-phenylenediamine, and the color assistant is hydrogen peroxide. Law.