JPH06102275A - Measuring method of steroid hormone - Google Patents

Abstract

PURPOSE:To allow accurate measurement of the concentration of steroid hormone in a sample by allowing coexistence of other steroid hormones in the sample thereby blocking bonding of steroid hormone to be measured and a binding protein. CONSTITUTION:Steroid hormone to be measured includes estradiol, estriol, testosterone, progesterone, cortisol, dihydroepiantrosterone sulfuric acid, and the like. Since these hormones bond with a binding protein in the blood, a steroid hormone other than that to be measured is added in order to block bonding. Such steroid hormone is added by 10-500ng/ml into the sample. When the sample is heated at 10-40 deg.C for 5 min-5 hours, bonding of hormone and binding protein can be blocked. Hormone is measured after anti-bonding reaction.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immunologically measuring a steroid hormone, which inhibits the binding of the steroid hormone to a binding protein and accurately measures the steroid hormone concentration.

[0002]

It is widely accepted that most of small molecules in blood are bound to a binding protein. For example, T3 and T4, which are thyroid hormones, have 99.9% and 99.5% bound to proteins in plasma. In cortisol, 95% is known to exist in association with corticosteroid-binding globulin, and in estradiol and testosterone, it is clear that it is associated with sex hormone-binding globulin. Has become. It is well known to measure the binding between these binding proteins and small molecules by releasing the target small molecule with a protein denaturant or a protein adsorbent. For example,
8-anilino-1-naphthalene sulfonic acid (AN
S), dilantin and the like. However, these inhibitors do not always work effectively.

Small molecules in serum samples exist in two forms, free and bound, and both are considered to be clinically important. In addition, it is important to measure the total concentration of estradiol, estriol, progesterone, etc. in the serum sample, which is the basis of clinical judgment. If necessary, pretreat the serum sample with an organic solvent,
It may be measured after releasing the small molecule from the binding protein. For example, removal of bound protein by dichloromethane or methanol, but nonspecific precipitation causes measurement error, and reliable accuracy and reproducibility remain a problem. This method is also very time consuming and involves the risk of handling organic solvents.

A conventionally known inhibitor is a glutamic acid solution in an immunoassay for cortisol (JP-A-53-53).
101521), and also 8-anilino-1-naphthalenesulfonic acid (ANS) in the method for analyzing cortisol.
Alternatively, a salt thereof (Japanese Patent Publication No. 61-12547) has been reported. ANS is known to bind to the hydrophobic region of proteins and is therefore believed to inhibit small molecule binding. In immunoassays for thyroid hormones, the inhibitors utilized are described in detail in US Pat. No. 3,911,096. However, these inhibitors
Since it affects all the proteins present in the reaction, it may induce unnecessary denaturation and inhibit the antigen-antibody reaction. Further, these inhibitors have an effective pH, and it is hard to say that they are effective in the vicinity of neutrality.

[0005]

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

[0006]

The present inventors have arrived at the present invention as a result of extensive studies on the above problems. That is, in the present invention, in the measurement of steroid hormones, a steroid hormone other than the measurement target is allowed to coexist in the sample, the binding between the measurement target hormone and the binding protein is inhibited, and then the measurement target hormone in the sample is directly measured. Is a method for measuring steroid hormones. The present invention will be described in detail below.

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

Since such a hormone is bound to the binding protein in blood, a steroid hormone other than the steroid hormone to be measured is added to inhibit the binding. Examples of the steroid hormone to be added include 5-dehydrotestosterone, pregnane (C21) derivative, 19-nortestosterone, 19-norprogesterone, androstenedione in addition to the above hormones. These steroid hormones added are preferably 10 to 500 n in the measurement sample.
g / ml, more preferably 50 to 300 ng / ml. And preferably, the temperature is 10-4
The reaction between 5 ° C. and the 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 5 hours. The binding inhibition reaction may be performed on the measurement sample in advance, and then the hormone may be measured, or the binding inhibition reaction and the hormone may be simultaneously measured.

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.

[0010]

According to the present invention, the concentration of steroid hormones in a sample can be accurately measured without extraction operation. Various types of steroid-binding proteins have been revealed. And the blood volume of men, women,
It depends on the pregnant woman. It is very useful to measure an accurate concentration of small molecules in the presence of binding proteins having different quantitative ratios, and the small molecules used in EIA and RIA can be used as a very effective means in clinical tests. It easily solves many requests for shortening of measurement time and development of automation, and its application range is wide.

[0011]

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) Reagent for measurement of estradiol Alkaline phosphatase-labeled estradiol solution Testosterone was adjusted to 50 ng / ml at 0.5%.
It was dissolved in a 50 mM Tris-hydrochloric acid buffer solution (pH 7.0) containing bovine serum albumin to obtain a diluted solution 1.

ANS was dissolved in 50 mM Tris-hydrochloric acid buffer (pH 7.0) containing 0.5% bovine serum albumin so that the dilution was 2.

50m containing 0.5% bovine serum albumin
M Tris-HCl buffer (pH 7.0) was used as diluent 3.

The alkaline phosphatase-labeled estradiol solution was treated with 0.0007 Abs at an absorbance of 280 nm.
Were added to each of the diluted solutions and mixed well. Estradiol antiserum Rabbit anti-estradiol antiserum was added to each diluted solution at 40,000 times. Estradiol standard solution Esterdiol is 0 (standard solution 1) in human serum containing no steroid, 48, 138, 460, 1900, 3
It was prepared by adding 950 pg / ml. Samples for addition recovery test Human serum was prepared, and 815 pg / ml, 1788 pg /
ml, and 4015 pg / ml estradiol were added and used to determine the amount recovered. (2) Measurement of Standard Solution Into 25 μl of the three kinds of alkaline phosphatase-labeled estradiol solution prepared in the above, 75 μl of each standard solution prepared in was dispensed with a micropipette. Next, add 50 μl of each antiserum solution prepared in
After mixing well with a vortex mixer, AIA12
00 (manufactured by Toso Co., Ltd.) at 37 ° C. for 40 minutes, and the rate of increase of the fluorescence intensity of 4-methylununveriferone by alkaline phosphatase was measured by a fluorometer. Each standard solution was measured 5 times.

Table 1 shows the relationship between each standard solution concentration and each binding rate (B / B ₀ ) when each dilution solution was used.
Shown in. The binding rate (B / B ₀ ) was calculated by the following formula.

B / B ₀ = (amount of increase in fluorescence of each standard solution) ÷
(Increase in fluorescence of standard solution 1) × 100 As is clear from the table, no significant difference was observed in the standard curve even when testosterone was added. It is considered that this is because the steroid-free human serum used for the preparation of the standard solution did not bind to estradiol due to the removal or denaturation of the estradiol binding protein during the desteroidal manipulation.

Table 1 Diluting solution 1 Diluting solution 2 Diluting solution 3 Standard solution concentration (Testosterone) (ANS) (Not added) (pg / ml) ―――――――― ―――――――――――― ―――――― Rate Bonding Rate Rate Bonding Rate Rate Bonding ―――――――――――――――――――――――――――――――――――― 0 45. 4 100 50. 6 100 44. 3 100 48 34. 1 75. 240. 279. 4 34. 277. 4 138 24. 6 54. 1 30. 0 59. 1 25. 3 57. 2 460 13. 2 28. 9 17. 0 33. 5 14. 0 31. 7 1900 4. 7 10. 4 6. 6 13. 1 5. 0 11. 4 3950 2. 8 6. 0 4. 1 2. 2 3. 1 7. 1 (3) 815 pg / ml of estradiol prepared in addition recovery test,
Samples with 1788 pg / ml and 4015 pg / ml added
Serum was added to each reaction reagent as in (2), and 3
Measured each time. Recovery rate is calculated by the following formula,
The results are summarized in Table 2.

Recovery rate = (measured value) ÷ (added value) × 100 As a result, it was revealed that the recovery of estradiol was improved by adding testosterone. Further, the effect was not observed in the diluted solution 2 to which the general inhibitor ANS was added, and the result was worse than that of the diluted solution 3 without addition. It is considered that the sample serum prepared in (1) had an estrone-binding protein, which bound to the estrone in the sample, and the binding was inhibited by testosterone.

Table 2 Addition amount Diluting solution 1 Diluting solution 2 Diluting solution 3 ――――― ―――――――― ―――――――― ―――――――― Measured value Recovery rate measurement Value Recovery Measured value Recovery pg / ml pg / ml% pg / ml% pg / ml% ―――――――――――――――――――――――――――― ――――――― 815 772 94.7 415 50.9 617 75.7 1788 1648 92.2 941 52.6 1435 80.3 4015 3619 90.1 2225 55.4 2834 70.6 Example 2 ( 1) Progesterone measurement reaction reagent Alkaline phosphatase labeled progesterone solution 0.5% cortisol to 100 ng / ml
It was dissolved in 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing bovine serum albumin to obtain a diluted solution 1.

50 mM Tris-hydrochloric acid buffer (pH 0.5%) containing no cortisol and containing 0.5% bovine serum albumin
7.5) was designated as dilution liquid 2.

Alkaline phosphatase labeled progesterone solution was added to 0.0007 Abs at 280 nm absorbance.
Were added to each of the diluted solutions and 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 steroid-free human serum at 0 (standard solution 1), 0.5, 2.0, 6.2, 20, 62.
It was prepared by adding it to ng / ml. Sample for addition recovery test Human serum was prepared, 0.400 ng / ml of progesterone was added, and it was used to determine the recovery amount. (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 each antiserum solution prepared in the above was added and mixed well with a vortex mixer, and then 3 with AIA1200 (manufactured by Toso Co., Ltd.).
The reaction was carried out at 7 ° C. for 40 minutes, and the rate of increase in the fluorescence intensity of 4-methylununbelliferone due to alkaline phosphatase was measured with a fluorometer. Each standard solution was measured 5 times.

Table 3 shows the relationship between the concentration of each standard solution and the binding rate (B / B ₀ ) when each diluted solution was used.
Note. The binding rate (B / B ₀ ) was calculated by the following formula.

B / B ₀ = (amount of increase in fluorescence of each standard solution) ÷
(Increase in fluorescence of standard solution 1) × 100 As is clear from the table, no significant difference was observed in the standard curve even when cortisol was added. This is considered to be because the steroid-free human serum used for the preparation of the standard solution did not bind to progesterone due to the removal or denaturation of the progesterone-binding protein during the desteroidal manipulation.

[0024] (3) Addition recovery test A human serum sample to which 0.40 ng / ml of progesterone prepared in the above was added was measured three times in the same manner as in (2). The recovery rate was calculated by the following calculation formula, and the results are summarized in Table 4.

Recovery rate = (measured value) ÷ (added value) × 100 As a result, it became clear that the recovery of progesterone was improved by adding cortisol. This is because there is a progesterone binding protein in the sample serum prepared in, and it binds to progesterone in the sample,
It is considered that the binding was inhibited by cortisol.

Table 4 Diluent 1 Diluent 2 Addition amount ――――――――― ――――――――― ng / ml Measured value Recovery rate Measured value Recovery rate ng / ml% ng / ml% ――――――――――――――――――――――――――― 0.400 0.407 102 0.111 27.9

Claims (2)

[Claims] 1. When measuring a steroid hormone, a steroid hormone other than the measurement target is allowed to coexist in the sample to inhibit the binding between the measurement target hormone and the binding protein, and
A method for measuring a steroid hormone, which comprises directly measuring a hormone to be measured in a sample. 2. The method according to claim 1, wherein the steroid hormone is estradiol, estriol, testosterone, progesterone, cortisol, or dehydroepiandrosterone sulfate.