JP2003028859A - Kit for differentiation of corpus luteum incompetence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means by which a progesterone amount in a human body is monitored carefully so as to surely differentiate corpus luteum incompetence. SOLUTION: Silvia is sampled at least once in two days in a luteal phase, the concentration of progesterone in the silvia is measured, and the corpus luteum incompetence is differentiated in such a way that the transition of the progesterone amount in the silvia for at least one week is used as an index. When the transition of the progesterone amount in the period is used as the index, 60 pg/ml is used as a threshold value in the concentration of the progesterone in the silvia during the measuring period.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for discriminating luteal dysfunction and a measuring kit therefor.

[0002]

Background Art Luteal dysfunction is one of female hormones.
Although the amount of progesterone secreted is remarkably reduced, it is a disease that develops as a symptom of infertility, shortening of the menstrual cycle, amenorrhea, etc. As a standard for differentiation, the amount of progesterone in blood is 10 nanograms / Less than or equal to ml is used as an index. However, the amount of progesterone secreted dynamically changes depending on the sexual cycle, so even in healthy women who do not have luteal dysfunction, the amount of progesterone in the blood sometimes falls below this level. It was hard to say that serious and serious illness could not be overlooked due to unnecessary administration of progesterone or misdiagnosis of luteal dysfunction. This is largely due to the fact that blood collection can only be performed in hospitals and that the blood collection itself cannot be performed frequently, which makes it impossible to monitor the secretion of progesterone in detail.

On the other hand, changes in the amount of progesterone secreted in saliva have never been monitored, nor has its involvement with luteal dysfunction been investigated. this is,
This is because it was generally believed that hormones had significantly lower concentrations in saliva than blood levels and were below the measurement limit. Further, it has not been reported that progesterone stably exists in saliva for a long time, and it was considered that there is no significance in measuring and using progesterone in saliva as an index.

[0004]

The present invention has been made under such circumstances, and by using saliva as a sample, the amount of progesterone in the human body is closely monitored to detect luteal dysfunction. An object of the present invention is to provide a means for surely discriminating between.

[0005]

[Means for Solving the Problems] In view of such a situation, the present inventors have made diligent research efforts in search of a means for precisely monitoring the amount of progesterone in the human body and for reliably distinguishing luteal dysfunction. As a result of repeating, at least 2 in the luteal phase
Saliva was collected once a day, the concentration of progesterone in the saliva was measured, and the change in the amount of progesterone in saliva over at least one week was used as an index to accurately identify luteal dysfunction. Heading, and came to complete the invention. That is, the present invention relates to the techniques described below. (1) A method for distinguishing luteal dysfunction, in which saliva is collected at least once every two days in the luteal phase, and the concentration of progesterone in the saliva is measured to measure the amount of progesterone in saliva for at least one week. A method for differentiating luteal dysfunction, which uses transition as an index. (2) Using the transition of the amount of progesterone in saliva over at least 2 weeks as an index, the concentration of progesterone in saliva during the measurement period is 60 picogram / ml.
Is used as the threshold value, and the method for differentiating luteal dysfunction according to (1). (3) The progesterone concentration in saliva is measured using an anti-progesterone antibody, a progesterone-binding peroxidase, a substrate of peroxidase, and an oxidant, the luteal dysfunction according to (1) or (2). Method for differentiating disease. (4) A kit for distinguishing luteal dysfunction, which comprises a well to which an anti-progesterone antibody is fixed, a progesterone-binding peroxidase, a substrate of peroxidase, and an oxidant as constituents. Hereinafter, the present invention will be described in detail centering on the embodiment.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Method for Differentiating Luteal Dysfunction of the Present Invention The method for differentiating luteal dysfunction of the present invention is to collect saliva at least once every two days in the luteal phase, and to obtain progesterone in the saliva. Is measured and the transition of the amount of progesterone in saliva over at least one week is used as an index. This is based on the following findings obtained as a result of the study by the present inventors.
That is, 1) reproducible measurement of the amount of progesterone in saliva is possible, and 2) progesterone is stably present in saliva, and its concentration hardly changes even when it is stored at room temperature for 14 days. There are two things that do not exist. However,
It is desirable to store frozen to prevent spoilage. In the discrimination method of the present invention, the concentration of progesterone in saliva can be measured by a commonly known method. As such a quantification method, for example, an antibody against progesterone is prepared, the antibody is labeled, and quantification is performed using this as an index. Such indicators include fluorescent labels, radiolabels, peroxidase labels and the like, and any of these can be used. Of these, a particularly suitable one is a peroxidase label whose handling and quantification can be carried out by an ordinary apparatus, and such a label can be produced by binding peroxidase to progesterone. That is, progesterone-binding peroxidase and salivary progesterone are allowed to competitively react with the immobilized anti-progesterone antibody, and the amount of unreacted progesterone-binding peroxidase is quantified by using the peroxidase substrate and oxidant. Thus, the amount of progesterone competitively present can be quantified. As a quantification using such a substrate of peroxidase and an oxide, hydrogen peroxide is used as a substrate of peroxidase, o-methylphenylenediamine and the like are added as an oxidizable substance, and the color developed by oxidation is measured. Therefore, quantification can be preferably exemplified.

(2) Discrimination method of the present invention The discrimination method of the present invention discriminates luteal dysfunction by following the change in the concentration of progesterone in saliva according to the above-mentioned measuring method. Following changes in concentration is to collect saliva at least once every two days in the luteal phase and follow salivary progesterone levels for at least one week. That is, to follow the progesterone concentration in saliva at least three times a week in the luteal phase. A more preferable form is to collect about 5 times a week for 2 weeks in the luteal phase. As shown in Examples below, in saliva, since progesterone is extremely stable, the subject brought back the saliva collection container by the required number of points, collected periodically, and subjected to quantitative analysis collectively, Such follow-up can be done easily. This is because the work of collecting saliva can be easily performed by anyone without a doctor, and thus the number of collection points can be increased in the discrimination method of the present invention unlike the conventional blood.
In monitoring progesterone in blood, the number of measurement points is reduced, so it cannot be concluded immediately that luteal dysfunction is present even if the amount of progesterone in blood is low. This is because progesterone, which is a sex hormone, changes largely and cyclically, and there is a possibility that the measurement point happened to fall during the time when progesterone was low. On the other hand, when the amount of progesterone in saliva, which is a feature of the identification method of the present invention, is used as an index, continuous sampling can be performed for a long period of time, and the cycle fluctuation itself can be captured. In order to perform such reliable differentiation of luteal dysfunction, saliva is collected at least once every two days in the luteal phase, preferably every day for at least one week, preferably for two weeks or more, and the amount of these progesterone is determined. The measurement can be preferably exemplified as the condition. In addition, as a criterion for distinguishing luteal dysfunction, it is preferable to use a threshold of 60 picogram / ml in the follow up of the concentration of progesterone in saliva. That is, when the progesterone concentration in saliva is lower than 60 picogram / ml at all the measuring points in the follow-up period, it is distinguished as luteal dysfunction.

[0008]

The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

<Example 1> According to the following method,
Prepare wells immobilized with anti-progesterone monoclonal antibody and progesterone-binding peroxidase,
Using this, it was examined whether there is a difference in salivary progesterone concentration between a person with a definite diagnosis of luteal dysfunction and a healthy person. (Preparation of mouse anti-progesterone monoclonal antibody)
The anti-progesterone antibody can be produced by a usual method. Namely, mouse, rat, guinea pig,
Immunize animals such as rabbits and sheep with Freund's complete or incomplete adjuvant to immunize them with progesterone bound to proteins such as bovine serum albumin as an antigen, and sera obtained from these animals to a protein A column. It can be obtained by fractionating the IgG fraction by affinity chromatography such as. Alternatively, a monoclonal antibody can be prepared from a hybridoma of immunized spleen cells and myeloma cells. Since many of these antibodies are already on the market, they can be purchased and used. These antibodies may be polyclonal antibodies or monoclonal antibodies, but antibodies having high sensitivity to progesterone and high specificity are preferable. (Adjustment of mouse anti-progesterone monoclonal antibody-immobilized wells) In each well of the plate, an antiserum obtained by immunizing other animals with the animal species that created the antibody as an antigen (for example, if an anti-mouse progesterone antibody, After coating with anti-mouse rabbit serum (RAM), anti-progesterone antibody is added to immobilize. The amount of antibody is not particularly limited as long as it is a detectable amount, and is adjusted depending on the specificity and sensitivity of the antibody, but in order to recognize a small amount of progesterone, it is preferably 0.05 to 50 ng per well,
0.1-10 ng is more preferable. (Preparation of Progesterone-Binding Peroxidase) Progesterone 11α-hemisuccinate and peroxidase are amide-bonded by a mixed acid anhydride method such as isobutylchlorocarbonate or a carbodiimide method, and purified by a gel filtration column or the like. The binding molar ratio of progesterone and peroxidase is appropriately adjusted depending on the sensitivity and specificity with the antibody,
To increase sensitivity, 5: 1 to 50: 1 is preferable, 1
More preferred is 0: 1 to 30: 1. (Measurement of Progesterone Concentration in Saliva) Using the above kit, the progesterone concentration in saliva was quantified by the absolute calibration curve method. That is, the saliva was incubated at 56 ° C. for 2 hours and centrifuged at 8000 g for 1 to 2 minutes.
l and 180 μl of progesterone-binding peroxidase solution
Was weighed into an anti-progesterone monoclonal antibody-immobilized well, the plate was sealed and mixed, and the mixture was incubated at 37 ° C for 1 hour, the specimen was fractionated by an immunoreaction, the liquid was removed, and the well was washed 3 times with a washing liquid. Washed. After adding 150 μl of color reagent to the washed wells and sealing, mix well, leave at room temperature (25 ± 2 ° C) for 30 minutes in the dark, add 50 μl of stop solution, seal, and mix for 1-2 minutes, then react. Stopped. 492 using a plate reader
The absorbance at nm was measured. The progesterone concentration in saliva was calculated from the absorbance from a calibration curve measured using a standard progesterone solution. (Group comparison) Table 1 shows a comparison of salivary progesterone concentrations between the luteal dysfunction group and the healthy group in the mid-luteal phase.
The unit of the numerical value is picogram / ml. From this, it can be seen that there is a clear difference in the progesterone concentration in saliva between the two groups, and that 60 picogram / ml can be set as the threshold value.

[0010]

[Table 1]

<Example 2> The stability of progesterone in saliva was examined. Saliva was stored at room temperature. Quantification was performed by the method of Example 1 using the kit of Example 1 above. The results are shown in Table 2. This table shows the stability (n = 3) under each storage condition. From this, it can be seen that progesterone is stably present in saliva, and therefore, saliva is periodically sampled and quantified at the same time, so that the concentration of progesterone can be followed.

[0012]

[Table 2]

<Example 3> The progesterone concentration in saliva of a person suspected of luteal dysfunction was followed from the blood progesterone concentration, and the authenticity thereof was examined. The measurement method was performed in the same manner as in Example 1, using the kit of Example 1. The follow results are shown in FIGS. From these results, it was revealed that these patients were suspected to have luteal dysfunction based on the blood concentration of progesterone, but this was simply due to low secretion of progesterone at the time of measurement, and no luteal dysfunction was observed. Became. That is, it can be seen that the differentiation of luteal dysfunction requires monitoring of the amount of progesterone at a plurality of points for a long period of one week or more.

<Example 4> On the other hand, contrary to Example 3, the concentration of progesterone in the blood was followed from the concentration of progesterone in the blood, and the true or false thereof was examined. . The measurement method was the same as in Example 3 except that the kit of Example 1 was used and the measurement was performed in the same manner as in Example 1. The follow results are shown in FIG. From this, it was revealed that luteal dysfunction was suspected in this case from the profile of blood progesterone concentration. Further, in the case of FIG. 5, it may be diagnosed that the luteal function is completely normal from the blood progesterone concentration measured by two blood collections, but from the monitoring of salivary progesterone concentration throughout the luteal phase, Suspected luteal shortening, luteal function evaluation
This suggests the importance of monitoring the amount of progesterone throughout the luteal phase rather than the measurement values at one or two points.

[0015]

Industrial Applicability According to the present invention, it is possible to provide a means for monitoring the amount of progesterone in the human body in detail and accurately distinguishing luteal dysfunction.

[Brief description of drawings]

FIG. 1 is a diagram showing a follow-up result of Example 3.

FIG. 2 is a diagram showing a follow-up result of Example 3.

FIG. 3 is a diagram showing a follow-up result of Example 3.

FIG. 4 is a diagram showing monitoring results of Example 4.

FIG. 5 is a diagram showing a monitoring result of Example 5.

Continued front page    (72) Inventor Akira Ito             1130-4 Kamekubo, Oi-cho, Iruma-gun, Saitama Stock             Company medicine (72) Inventor Satoshi Miyata             Stock exchange, 2-8-16 Funato, Itabashi-ku, Tokyo             In-house medicine (72) Inventor Yoshihisa Fukai             1130-4 Kamekubo, Oi-cho, Iruma-gun, Saitama Stock             Company medicine F term (reference) 2G045 AA25 CA25 CB07 DA54 DA77                       DB21 FB03 FB07 FB11 GC10                       JA01 JA06                 4B063 QA01 QA19 QQ03 QQ22 QR02                       QR41 QR50 QR81 QX01

Claims (4)

[Claims] 1. A method for distinguishing luteal dysfunction, comprising collecting saliva at least once every two days in the luteal phase, measuring the concentration of progesterone in the saliva, and progesterone in saliva for at least one week. A method for distinguishing luteal dysfunction, which is characterized by using changes in amount as an index. 2. The concentration of progesterone in saliva during the measurement period is defined as a threshold value of 60 picogram / ml, using the transition of the amount of progesterone in saliva over at least 2 weeks as an index. The method for differentiating luteal dysfunction according to claim 1. 3. The luteal dysfunction according to claim 1 or 2, wherein the progesterone concentration in saliva is measured using an anti-progesterone antibody, a progesterone-binding peroxidase, a substrate of peroxidase and an oxidant. Method for differentiating disease. 4. A kit for distinguishing luteal dysfunction, which comprises a well to which an anti-progesterone antibody has been fixed, a progesterone-binding peroxidase, a substrate of peroxidase, and an oxide.