JPH0634636A - Measuring method for steroid hormone - Google Patents

Abstract

PURPOSE:To exactly measure hormone concentration of a specimen by checking the combination of steroid hormone and binding protein with salicylic acid or its salt. CONSTITUTION:Androgen, estrogen and the like, for instance, estradiol, oestriol, progesterone and the like are mentioned as steroid hormone to be measured. Since a such hormone is bound to binding protein in blood, salicylic acid or its salt is added because its combination is checked. The salt of salicylic acid, for instance, sodium salicylate, calcium salicylate and the like are mentioned. These salicylic acid or its salt is preferably 0.1-2 % in a specimen to be measured and added so as to be furthermore preferably 0.2-1.0%. And it is reacted at about 4-5 deg.C for about 5 minutes-several hours in proportion to temperature and the combination of hormone and binding protein is checked. The check reaction may as well be performed before hormone measurement, at the same time as measurement or in either may.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a steroid hormone, which comprises accurately measuring the concentration of a steroid hormone in a sample by inhibiting the binding of the steroid hormone to a binding protein.

[0002]

2. Description of the Related Art It is widely accepted that most of small molecules such as hormones in blood are bound to a binding protein. For example, T3 and T4, which are thyroid hormones
Are bound to protein in plasma at 99.9% and 99.5%, respectively. In cortisol, 95% is known to exist in association with corticosteroid-binding globulin, and also in estradiol and testosterone, it may be associated with sex hormone-binding globulin. It is clear.

Hormones in serum samples exist in two forms, free and bound, and are considered to be clinically important. In addition, Estradiol and Estrio-
It is important to measure the total concentration of serum, progesterone, cortisol, etc. in a serum sample, which is the basis of clinical judgment. If necessary, the serum sample may be pretreated with an organic solvent to release the hormone from the bound protein and then measured.

For example, removal of the bound protein by dichloromethane or methanol may be mentioned, but non-specific precipitation causes measurement errors, and reliable accuracy and reproducibility remain a problem. Also, this method is very time consuming and involves the risk of handling organic solvents.

Cortisol is a known inhibitor.
Glutamic acid solution in the immunoassay method
No. 101521), there is also 8-anilino-1-naphthalenesulfonic acid (ANS) or a salt thereof (Japanese Patent Publication No. 61-12547) in the method for analyzing cortisol. ANS is known to bind to the hydrophobic region of proteins and is therefore believed to inhibit hormone binding. In immunoassays for thyroid hormones, the inhibitors utilized are described in detail in USP 3,911,096.

[0006]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a method for accurately measuring the hormone concentration in a sample by measuring the hormone bound to the binding protein in the free form when measuring the steroid hormone. To do.

[0007]

In view of the above, the present inventors have arrived at the present invention as a result of extensive studies on inhibitors for measuring hormones. That is, the present invention is a method for measuring a steroid hormone, which comprises measuring the steroid hormone by inhibiting the binding between the steroid hormone and the binding protein with salicylic acid or a salt thereof. Hereinafter, the present invention will be described in detail.

The steroid hormones measured in the present invention include androgens, estrogens, gestagens,
There are corticoids, etc. Specifically, Estradio-
Le, estriol, progesterone, cortisol,
Examples include testosterone, dehydroepiandrosterone sulfate, and the like.

Since such a hormone is bound to the binding protein in blood, salicylic acid or its salt is added to inhibit the binding. Examples of salicylic acid salts include sodium salicylate, calcium salicylate, ammonium salicylate, and the like. These salicylic acids or salts thereof are preferably 0.1-2.0%, more preferably 0.2-1.0% in the measurement sample.
To be added. And preferably, the temperature is 4-5
The reaction between 0 ° C. and time depends on the temperature, but the reaction between the hormone and the binding protein can be inhibited by reacting for about 5 minutes to several hours. The binding inhibition reaction may be performed on the measurement sample in advance, and then the hormone may be measured, or the hormone may be measured at the same time.

The method for measuring the hormone is not particularly limited, but an immune reaction is preferable in terms of measurement sensitivity. Although there are various measuring principles for the immune reaction, the measuring method is not limited to a specific reaction, and examples thereof include a sandwich method using a monoclonal antibody or a polyclonal antibody, a competitive method, and an agglutination method. Further, a label may be used for detecting an immune reaction, and for example, a radioisotope, an enzyme, a fluorescent substance or the like can be used.

[0011]

According to the present invention, directly without extraction operation,
It is possible to accurately measure the hormone concentration in a sample. This is a solution to many requests for shortening the measurement time of hormones used in EIA and RIA, which is a very effective means in clinical examinations today, and development to automation, and its application range is wide.

[0012]

EXAMPLES Examples will be shown below for illustrating the present invention further in detail, but the present invention is not limited to these examples.

Example 1 (1) Cortisol measuring reaction reagent Alkaline phosphatase labeled cortisol solution Sodium salicylate was added to 0, 0.3, 0.5, 0.8,
It was dissolved in 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 0.5% bovine serum albumin so as to be 1.0%, and each was used as a diluting solution.

Alkaline phosphatase labeled cortisol
Solution was added to each dilution so that the absorbance was 0.001 Abs at 280 nm and mixed well. Cortisol antiserum Rabbit anti-cortisol antiserum was added 4000 times to each diluted solution. Cortisol standard solution Cortisol was added to human steroid-free human serum at 0, 2.5, 6.5, 14.0, 36.7 and 80.3, respectively.
A standard solution was prepared by adding and adjusting the concentration to be μg / dl. Unknown cortisol sample Two sera containing an unknown amount of cortisol were prepared. (2) Measurement of standard solution Alkaline phosphatase-labeled cortisol prepared in
10 μl of the standard solution of each concentration was dispensed into 50 μl of the solution with a micropipette. Next, 90 μl of the antiserum solution prepared in the above was added, and further solid-phased anti-rabbit antiserum was added. After thoroughly mixing with a vortex mixer, AI
A1200 (manufactured by Toso Co., Ltd.) was allowed to react at 37 ° C. for 40 minutes, and the rate of increase in the fluorescence intensity of 4-methylilunbelliferone due to alkaline phosphatase was measured with a fluorometer. Each standard solution was measured 5 times and the average value was calculated. (3) From the results of (2), the decomposition rate of the substrate (nmol / s
ec) was determined, and the ratio (B / Bo × 100) of the fluorescence increase amount (B) of each standard solution to the fluorescence increase amount (Bo) of the standard solution having a cortisol concentration of 0 μg / dl was calculated. The results are shown in Table 1. As in the table, a standard curve was obtained for samples of known concentration.

Table 1 Cortisol sodium salicylate (concentration%) μg / dl 0 0.3 0.5 Decomposition rate B / Bo decomposition rate B / Bo decomposition rate B / Bo ───────────── ─────────────────────── 0 50.3 100 52.4 100 51.3 100 2.5 23.1 45.9 23.7 45. 2 24.4 47.6 6.5 15.7 31.2 15.7 29.9 16.4 32.1 14.0 11.4 22.7 11.8 22.4 11.3 21.9 36 6.7 6.7 13.4 7.1 13.5 7.1 7.1 13.9 80.3 4.6 9.2 4.7 4.7 9.0 5.0 9.8 Cortisol sodium salicylate (concentration%) μg / dl 0.8 1.0 Decomposition rate B / Bo Decomposition rate B / Bo ───────────── ─────────────── 0 50.3 100 50.1 100 2.5 23.3 46.4 23.8 47.4 6.5 15.6 31.0 14. 9 29.6 14.0 11.3 22.4 10.9 21.9 36.7 7.0 7.0 14.0 6.9 13.7 80.3 5.0 10.0 10.0 4.9 9.8 ( 4) Measurement of unknown sample Serum 1 and 14.0 μg / dl obtained by adding 2.9 μg / dl of cortisol to serum containing an unknown amount of cortisol.
10 μl of each of the serums 2 to which was added was mixed well with 140 μl of the reaction reagent having each salicylic acid concentration, and the measurement was performed in the same manner as in Example 1.

The results are shown in Table 2 using the ratio (%) of the added amount of cortisol calculated from the measured value to the amount of actually added cortisol as the recovery rate.

Table 2 Sodium salicylate concentration (%) 0 0.3 0.5 0.8 1.0 1.0 ─────────────────────────── ──── Serum 1 47.5 82.0 108 95.5 114 Serum 2 78.8 87.3 94.0 94.0 94.0 As is clear from the results in Table 2, sodium salicylate 0.3% was added. % Of cortisol in serum samples
It was possible to accurately measure the amount of solder.

Example 2 (1) Progesterone measurement reaction reagent Alkaline phosphatase labeled progesterone solution 50 mM Tris containing 0.5% bovine serum albumin so that sodium salicylate becomes 0, 0.2 and 0.4%. -Dissolved in hydrochloric acid buffer solution (pH 7.5) and used each as a diluent.

The alkaline phosphatase-labeled progesterone solution was added to each dilution so that the absorbance at 280 nm was 0.001 Abs, and they were mixed well. Progesterone antiserum Rabbit anti-progesterone antiserum was added to each diluted solution at 50,000 times. Progesterone standard solution Progesterone was added to human serum containing no steroid to 0,
It was prepared by adding to 0.5, 2, 10, 30, 60 ng / ml. Unknown progesterone sample One serum containing an unknown amount of progesterone was prepared. (2) Measurement of Standard Solution Into 25 μl of the alkaline phosphatase-labeled progesterone solution prepared in the above, 75 μl of each standard solution was dispensed with a micropipette. Next, 50 μl of the antiserum solution prepared in the above was added, and further solid-phased anti-rabbit antiserum was added. After mixing well with a vortex mixer, AIA
The reaction was performed at 1200 (manufactured by Toso Co., Ltd.) at 37 ° C. for 40 minutes, and the rate of increase in the fluorescence intensity of 4-methylununveriferone due to alkaline phosphatase was measured with a fluorometer. The measurement was performed 5 times and the average value was calculated. (3) From the results of (2), the decomposition rate of the substrate (nM / se
c) was calculated, and the ratio (B / Bo × 100) of the fluorescence increase amount (B) of each standard solution to the fluorescence increase amount (Bo) of the standard solution having a progesterone concentration of 0 ng / ml was calculated.
Shown in.

Table 3 Progesterone sodium salicylate (concentration%) ng / ml 0 0.2 0.4 Degradation rate B / Bo Degradation rate B / Bo Degradation rate B / Bo ───────────── ─────────────────────── 0 85.6 100 80.2 100 86.9 100 0.1 81.2 94.8 75.6 94. 2 82.4 94.8 1.0 69.8 81.5 60.9 76.0 69.0 79.4 3.9 44.5 51.9 39.6 49.3 41.8 48.1 9 2.8 27.3 31.8 24.7 30.8 25.4 29.2 41.2 10.1 10.1 11.8 8.7 10.9 9.5 10.9 Samples with known concentrations A standard curve for was obtained. (4) Measurement of unknown sample 0.40 was added to serum containing unknown amount of progesterone.
ng / ml progesterone was added. This sample 75
μl was added to the reaction reagent in the same manner as in (2), and measurement was performed. The measurement was performed 3 times and the average value was calculated. The results are shown in Table 4 using the ratio (%) of the added amount of progesterone calculated from the measured value to the amount of actually added progesterone as the recovery rate.

Table 4 Sodium salicylate concentration (%) 0 0.2 0.4 ───────────────────────────── Progesterone 0. 40 ng / ml addition 35.9 79.2 126 It became clear from Table 4 that the recovery of progesterone was improved depending on the salicylic acid concentration, and by adding sodium salicylate in an amount of 0.2% or more, the serum sample It was possible to accurately measure the amount of progesterone.

Claims (3)

[Claims] 1. A method for measuring a steroid hormone, which comprises measuring the steroid hormone by inhibiting the binding between the steroid hormone and a binding protein with salicylic acid or a salt thereof. 2. The steroid hormone is estradiol,
The assay method according to claim 1, which is estrol, progesterone, cortisol, testosterone, or dehydroepiandrosterone sulfate. 3. The salicylic acid or its salt concentration is 0.1%.
The measuring method according to claim 1 or 2, which is not less than 2.0%.