JP6934587B2 - Method for measuring steroid hormones in samples - Google Patents

Description

The present invention relates to a method for measuring a steroid hormone in a sample, a reagent for measurement, and a kit for measurement.

Steroid hormones are hormones having a molecular weight of 300 to 400 and having a steroid skeleton, and are classified into sex hormones, glucocorticoids, mineralocorticoids, and the like based on their functions. For clinical studies of steroid hormones, methods for measuring the amount of steroid hormones in vivo have been studied, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS). Methods, immunological measurement methods, etc. have been developed. Although the GC-MS method and the LC-MS method have high analytical ability, there are problems that the analyzer used for these analyzes is expensive and complicated operations such as concentration operation and extraction operation are required. .. On the other hand, immunological measurement methods are often used in clinical research because they can measure steroid hormones easily and in a short time.

As an immunological measurement method, a sandwich method, a competitive method, etc. are known, but usually, when a polymer such as a protein is targeted for measurement, the sandwich method is used, and a small molecule compound such as a steroid hormone is used. Is used as a measurement target, the competitive method is used. However, in the measurement of steroid hormones by the competitive method, the sensitivity and specificity are often increased due to the "cross-reactivity" in which the antibody used reacts with steroid hormones other than the substance to be measured having a structure similar to that of the substance to be measured. It becomes a problem. So far, in order to improve sensitivity, an antibody obtained by immunizing an animal with a low molecular weight compound bound to a carrier protein via a linker and a position different from the linker binding position in the low molecular weight compound bound to the carrier protein. A method for measuring the low molecular weight compound using a low molecular weight compound bonded to a labeling substance via a linker has been reported (see Patent Document 1).

Aldosterone is a mineral steroid hormone with a molecular weight of about 360 secreted from the globular layer belonging to the outer layer of the adrenal cortex, and its secretion is mainly dependent on the renin-angiotensin-aldosterone system. Aldosterone is a hormone that is crucial for maintaining electrolyte homeostasis, circulating blood volume, and blood pressure, and acts on mineral corticoid receptors in the distal convoluted tubules of the kidney to promote water reabsorption as well as sodium reabsorption. It has the function of raising blood pressure.

Aldosterone is used for differential diagnosis of primary aldosteronism, Bartter syndrome, Liddle's syndrome, 17α-hydroxylase deficiency in the adrenal gland, hydroxylase deficiency such as 11β-hydroxylase deficiency, and selective hypoaldosteronism. Has been done. In addition, in the guidelines of the Japanese Society of Endocrinology and the Japanese Society of Hypertension, as a screening method for primary aldosterone disease, the renin activity (PRA) or renin concentration (Active Renin Concentration) of the aldosterone concentration (PAC) is used. It is recommended to measure the Aldosterone to Renin Ratio (ARR), which is the ratio to: ARC).

LC-MS method, immunological measurement method and the like are known as aldosterone measurement methods, and radioimmunoassay method (RIA method) and enzyme immunoassay method (EIA method) are known as immunological measurement methods. (See Patent Document 2). However, when measuring aldosterone in plasma of patients with chronic renal failure by the RIA method, the steroid hormone reacts with the anti-aldosterone antibody because a large amount of steroid hormone having a structure similar to that of aldosterone is present in the sample. It has been reported that accurate measured values cannot be obtained due to the influence of cross-reactivity (see Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2013-03448 Japanese Unexamined Patent Publication No. 2-057976

J Lab. Clin. Med., Vol.114 (3), pp.294-300 (1989).

An object of the present invention is to provide a method, a reagent, and a kit that enable accurate measurement of a steroid hormone in a sample without being affected by a cross-reactive substance having a structure similar to that of the steroid hormone to be measured. To do.

The present inventors have diligently studied to solve this problem, and in the measurement by the competitive method of steroid hormones in a sample, the antigen-antibody reaction contains a buffer having a specific structure and a specific surfactant. We have found that cross-reactivity is suppressed and accurate measurement of steroid hormones is possible by performing in an aqueous medium, and completed the present invention. That is, the present invention relates to the following [1] to [23].

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤と、非イオン性界面活性剤、陰イオン性界面活性剤、及び、両性界面活性剤からなる群より選ばれる少なくとも１種の界面活性剤とを含有する水性媒体中で反応させて、抗ステロイドホルモン抗体と標識化競合物質との免疫複合体を生成させ、生成した当該免疫複合体中の標識を測定することを特徴とする、検体中のステロイドホルモンの測定方法。
［２］ 検体と抗ステロイドホルモン抗体とを反応させた後、当該反応の反応液に標識化競合物質を添加する、［１］記載の測定方法。
［３］ 抗ステロイドホルモン抗体が、不溶性担体に固定化されている、［１］又は［２］記載の測定方法。
［４］ 検体と、標識化抗ステロイドホルモン抗体及び競合物質とを、一般式（Ｉ）

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤と、非イオン性界面活性剤、陰イオン性界面活性剤、及び、両性界面活性剤からなる群より選ばれる少なくとも１種の界面活性剤とを含有する水性媒体中で反応させて、標識化抗ステロイドホルモン抗体と競合物質との免疫複合体を生成させ、生成した当該免疫複合体中の標識を測定することを特徴とする、検体中のステロイドホルモンの測定方法。
［５］ 検体と標識化抗ステロイドホルモン抗体とを反応させた後、当該反応の反応液に競合物質を添加する、［４］記載の測定方法。
［６］ 競合物質が、不溶性担体に固定化されている、［４］又は［５］記載の測定方法。
［７］ 緩衝剤が、２−モルホリノエタンスルホン酸（ＭＥＳ）、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）、及びビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）からなる群から選ばれる緩衝剤である、［１］〜［６］のいずれかに記載の測定方法。
［８］ ステロイドホルモンが、鉱質コルチコイドである、［１］〜［７］のいずれかに記載の測定方法。
［９］ 鉱質コルチコイドが、アルドステロンである、［８］記載の測定方法。 [1] The sample and the anti-steroid hormone antibody and the labeled competing substance are represented by the general formula (I).

(In the formula, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a buffer having a structure, a nonionic surfactant, an anionic surfactant, and The reaction was carried out in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to generate an immune complex of an antisteroid hormone antibody and a labeled competing substance. A method for measuring a steroid hormone in a sample, which comprises measuring a label in an immune complex.
[2] The measuring method according to [1], wherein after reacting the sample with an anti-steroid hormone antibody, a labeled competing substance is added to the reaction solution of the reaction.
[3] The measuring method according to [1] or [2], wherein the anti-steroid hormone antibody is immobilized on an insoluble carrier.
[4] The sample and the labeled anti-steroid hormone antibody and the competing substance are represented by the general formula (I).

(In the formula, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a buffer having a structure, a nonionic surfactant, an anionic surfactant, and The reaction was carried out in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to generate an immune complex of a labeled antisteroid hormone antibody and a competing substance. A method for measuring a steroid hormone in a sample, which comprises measuring a label in an immune complex.
[5] The measuring method according to [4], wherein after reacting the sample with a labeled anti-steroid hormone antibody, a competing substance is added to the reaction solution of the reaction.
[6] The measuring method according to [4] or [5], wherein the competing substance is immobilized on an insoluble carrier.
[7] The buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N. , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N'-(2-sulfoethyl) piperazin (HEPES), and bis (2-hydroxyethyl) iminotris The measuring method according to any one of [1] to [6], which is a buffer selected from the group consisting of (hydroxymethyl) methane (Bis-Tris).
[8] The measuring method according to any one of [1] to [7], wherein the steroid hormone is a mineralocorticoid.
[9] The measuring method according to [8], wherein the mineralocorticoid is aldosterone.

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤、標識化競合物質、並びに、抗ステロイドホルモン抗体を含む、検体中のステロイドホルモンの測定用試薬。
［１１］ 抗ステロイドホルモン抗体が、不溶性担体に固定化されている、［１０］記載の試薬。
［１２］ 非イオン性界面活性剤、陰イオン性界面活性剤、及び、両性界面活性剤からなる群より選ばれる少なくとも１種の界面活性剤、一般式（Ｉ）

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤、標識化抗ステロイドホルモン抗体、並びに、競合物質を含む、検体中のステロイドホルモンの測定用試薬。
［１３］ 競合物質が、不溶性担体に固定化されている、［１２］記載の試薬。
［１４］ 緩衝剤が、２−モルホリノエタンスルホン酸（ＭＥＳ）、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）、及びビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）からなる群から選ばれる緩衝剤である、［１０］〜［１３］のいずれかに記載の試薬。
［１５］ ステロイドホルモンが、鉱質コルチコイドである、［１０］〜［１４］のいずれかに記載の試薬。
［１６］ 鉱質コルチコイドが、アルドステロンである、［１５］記載の試薬。 [10] At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, general formula (I).

(In the formula, X represents a hydroxyl group or a sulfo group, n represents an integer of 2 to 5), and the sample contains a buffer having a structure represented by a buffer, a labeling competitor, and an antisteroid hormone antibody. Reagent for measuring steroid hormones.
[11] The reagent according to [10], wherein the anti-steroid hormone antibody is immobilized on an insoluble carrier.
[12] At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, general formula (I).

(In the formula, X represents a hydroxyl group or a sulfo group, n represents an integer of 2 to 5), and the sample contains a buffer having a structure represented by a buffer, a labeled antisteroid hormone antibody, and a competing substance. Reagent for measuring steroid hormones.
[13] The reagent according to [12], wherein the competing substance is immobilized on an insoluble carrier.
[14] The buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N. , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N'-(2-sulfoethyl) piperazin (HEPES), and bis (2-hydroxyethyl) iminotris The reagent according to any one of [10] to [13], which is a buffer selected from the group consisting of (hydroxymethyl) methane (Bis-Tris).
[15] The reagent according to any one of [10] to [14], wherein the steroid hormone is a mineralocorticoid.
[16] The reagent according to [15], wherein the mineralocorticoid is aldosterone.

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤のそれぞれが、第１試薬、第２試薬のいずれか又は両方に含まれる、検体中のステロイドホルモンの測定用キット。
［１８］ 抗ステロイドホルモン抗体が、不溶性担体に固定化されている、［１７］記載のキット。
［１９］ 標識化抗ステロイドホルモン抗体を含む第１試薬と、競合物質を含む第２試薬とを含有し、非イオン性界面活性剤、陰イオン性界面活性剤、及び、両性界面活性剤からなる群より選ばれる少なくとも１種の界面活性剤と、一般式（Ｉ）

（式中、Ｘは水酸基又はスルホ基を表し、ｎは２〜５の整数を表す）で表される構造を有する緩衝剤のそれぞれが、第１試薬、第２試薬のいずれか又は両方に含まれる、検体中のステロイドホルモンの測定用キット。
［２０］ 競合物質が、不溶性担体に固定化されている、［１９］記載のキット。
［２１］ 緩衝剤が、２−モルホリノエタンスルホン酸（ＭＥＳ）、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）、及びビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）からなる群から選ばれる緩衝剤である、［１７］〜［２０］のいずれかに記載のキット。
［２２］ ステロイドホルモンが、鉱質コルチコイドである、［１７］〜［２１］のいずれかに記載のキット。
［２３］ 鉱質コルチコイドが、アルドステロンである、［２２］記載のキット。 [17] It contains a first reagent containing an antisteroid hormone antibody and a second reagent containing a labeling competitor, and comprises a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. At least one surfactant selected from the group and the general formula (I)

Each of the buffers having a structure represented by (X represents a hydroxyl group or a sulfo group and n represents an integer of 2 to 5) is contained in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in samples.
[18] The kit according to [17], wherein the anti-steroid hormone antibody is immobilized on an insoluble carrier.
[19] It contains a first reagent containing a labeled antisteroid hormone antibody and a second reagent containing a competing substance, and comprises a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. At least one surfactant selected from the group and the general formula (I)

Each of the buffers having a structure represented by (X represents a hydroxyl group or a sulfo group and n represents an integer of 2 to 5) is contained in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in samples.
[20] The kit according to [19], wherein the competing substance is immobilized on an insoluble carrier.
[21] The buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N. , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N'-(2-sulfoethyl) piperazin (HEPES), and bis (2-hydroxyethyl) iminotris The kit according to any one of [17] to [20], which is a buffer selected from the group consisting of (hydroxymethyl) methane (Bis-Tris).
[22] The kit according to any one of [17] to [21], wherein the steroid hormone is a mineralocorticoid.
[23] The kit according to [22], wherein the mineralocorticoid is aldosterone.

INDUSTRIAL APPLICABILITY The present invention provides methods, reagents, and kits that enable accurate measurement of steroid hormones in a sample without being affected by cross-reactive substances.

(Measuring method)
In the method for measuring a steroid hormone in a sample of the present invention, a sample, an anti-steroid hormone antibody and a labeled competing substance are used in the general formula (I).

(In the formula, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5) and a buffer having a structure represented by the buffer [hereinafter referred to as buffer (I)] and a nonionic surfactant. , Anionic surfactants, and at least one surfactant selected from the group consisting of amphoteric surfactants. The method is characterized in that a complex is generated and the label in the generated immune complex is measured.

As one aspect (aspect 1) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] The sample and the anti-steroid hormone antibody and the labeled competing substance are selected from the group consisting of the buffer (I), the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant. Reaction in an aqueous medium containing at least one surfactant to produce an immune complex of an antisteroid hormone antibody and a steroid hormone, and an immune complex of an antisteroid hormone antibody and a labeled competitor. Process to make;
[2] A step of measuring a labeling substance in an immune complex of an anti-steroid hormone antibody produced in step [1] and a labeling competitor;
[3] A step of performing the above steps [1] and [2] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[4] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [3] and the measured value of the labeling substance measured in the step [2].

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but it is preferable that it is immobilized.

As another aspect (aspect 2) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] At least one type of surfactant selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an amphoteric tenside agent in a sample and an antisteroid hormone antibody. Step of reacting with an aqueous medium containing an agent;
[2] A step of adding a labeling competing substance to the reaction solution of the step [1];
[3] A step of measuring a labeling substance in an immune complex of an anti-steroid hormone antibody produced in step [2] and a labeling competitor;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

As another aspect (aspect 3) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] A step of reacting a sample and an anti-steroid hormone antibody in an aqueous medium containing a buffer (I);
[2] At least one surfactant selected from the group consisting of a labeled competing substance, a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant in the reaction solution of the step [1]. Step of adding agent;
[3] A step of measuring a labeling substance in an immune complex of an anti-steroid hormone antibody produced in step [2] and a labeling competitor;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

As another aspect (aspect 4) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] An aqueous medium containing a sample and an anti-steroid hormone antibody containing at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. The process of reacting inside;
[2] A step of adding a labeling competing substance and a buffer (I) to the reaction solution of the step [1];
[3] A step of measuring a labeling substance in an immune complex of an anti-steroid hormone antibody produced in step [2] and a labeling competitor;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

Furthermore, as another aspect (aspect 5) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] A step of reacting a sample with an anti-steroid hormone antibody in an aqueous medium;
[2] The reaction solution of step [1] is selected from the group consisting of a labeling competitor, a buffer (I), and a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. Step of adding at least one surfactant;
[3] A step of measuring a labeling substance in an immune complex of an anti-steroid hormone antibody produced in step [2] and a labeling competitor;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

When the anti-steroid hormone antibody is immobilized on an insoluble carrier, the insoluble carrier on which the anti-steroid hormone antibody is immobilized may be produced in the reaction solution of the antigen-antibody reaction, in which case, a set of affinity substances. An anti-steroid hormone antibody by reacting an anti-steroid hormone antibody to which one of the above (A) is bound and an insoluble carrier to which the other (a) of a set of affinity substances is bound in the reaction solution of the antigen-antibody reaction. An insoluble carrier on which is immobilized can be produced in the reaction solution of the antigen-antibody reaction. Examples of the combination of Aa include the following combinations and the like.
・ Combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
・ Combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
-A combination of the Fc region of an anti-steroid hormone antibody and an antibody that binds to the Fc region.

Reaction of the sample with the anti-steroid hormone antibody in the step [1] in the first aspect and the steps [1] and [2] in the second to fifth aspects, and the reaction of the labeling competitor with the anti-steroid hormone antibody. The temperature is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, preferably 2 minutes to 1 hour.

In the step [1] of the first to fifth aspects, the sample can be pretreated with an aqueous medium described later and then subjected to the reaction. Examples of the pretreatment include dilution, solubilization, denaturation and the like. The aqueous medium may contain metal ions, sugars, preservatives, proteins and the like, which will be described later. The temperature at which the sample is pretreated with an aqueous medium is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and 20 to 20 to. 40 ° C is particularly preferred. The time for pretreating the sample with an aqueous medium is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, preferably 2 minutes to 1 hour.

The method for measuring the labeling substance in the step [2] in the first aspect and the step [3] in the second to fifth aspects is not particularly limited as long as it is a method capable of measuring the steroid hormone of the present invention. The method and the like can be mentioned.

The method for measuring a steroid hormone in a sample of the present invention also comprises using a sample, a labeled antisteroid hormone antibody, and a competing substance as a buffer (I), a nonionic surfactant, and an anionic surfactant. , And react in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to generate and produce an immune complex of a labeled antisteroid hormone antibody with a competitor. This method is characterized by measuring a labeling substance in the immune complex.

As one aspect (aspect 6) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] A sample, a labeled antisteroid hormone antibody and a competing substance are selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. An immune complex of a labeled anti-steroid hormone antibody and a steroid hormone and an immune complex of a labeled anti-steroid hormone antibody and a competing substance are reacted in an aqueous medium containing at least one of the surfactants. The process of generating a body;
[2] A step of measuring a labeled substance in an immune complex of a labeled anti-steroid hormone antibody produced in step [1] and a competing substance;
[3] A step of performing the above steps [1] and [2] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[4] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [3] and the measured value of the labeling substance measured in the step [2].

The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized.

As another aspect (aspect 7) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] At least one selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant in a sample and a labeled antisteroid hormone antibody. Step of reacting in an aqueous medium containing the above-mentioned surfactant;
[2] A step of adding a competing substance to the reaction solution of the step [1];
[3] A step of measuring a labeled substance in an immune complex of a labeled anti-steroid hormone antibody produced in step [2] and a competing substance;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized.

As another aspect (aspect 8) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] A step of reacting a sample with a labeled anti-steroid hormone antibody in an aqueous medium containing a buffer (I);
[2] The reaction solution of step [1] contains a competing substance and at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. Step of adding;
[3] A step of measuring a labeled substance in an immune complex of a labeled anti-steroid hormone antibody produced in step [2] and a competing substance;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized.

As another aspect (aspect 9) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] The sample and the labeled antisteroid hormone antibody contain at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. The process of reacting in an aqueous medium;
[2] A step of adding a competing substance and a buffer (I) to the reaction solution of the step [1];
[3] A step of measuring a labeled substance in an immune complex of a labeled anti-steroid hormone antibody produced in step [2] and a competing substance;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized.

Furthermore, as another aspect (aspect 10) of the method for measuring a steroid hormone of the present invention, for example, a method including the following steps can be mentioned.
[1] A step of reacting a sample with a labeled anti-steroid hormone antibody in an aqueous medium;
[2] At least selected from the group consisting of a competing substance, a buffer (I), and a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant in the reaction solution of the step [1]. Step of adding one type of surfactant;
[3] A step of measuring a labeled substance in an immune complex of a labeled anti-steroid hormone antibody produced in step [2] and a competing substance;
[4] A step of performing the above steps [1] to [3] using a known concentration of steroid hormone instead of a sample to create a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[5] A step of determining the steroid hormone concentration in the sample from the calibration curve prepared in the step [4] and the measured value of the labeling substance measured in the step [3].

The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized.

When the competing substance is immobilized on an insoluble carrier, the insoluble carrier on which the competing substance is immobilized may be produced in the reaction solution of the antigen-antibody reaction, in which case one of a set of affinity substances (B). ) And the insoluble carrier to which the other (b) of the other affinity substance is bound are reacted in the reaction solution of the antigen-antibody reaction to obtain an insoluble carrier on which the competing substance is immobilized. It can be produced in the reaction solution of the antigen-antibody reaction. Examples of the combination of BB include the following combinations and the like.
・ Combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-A combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin.

Reaction of the sample with the labeled anti-steroid hormone antibody in steps [1] in Aspect 6 and steps [1] and [2] in Aspects 7 to 10, and a competitor and a labeled anti-steroid hormone antibody. The reaction temperature of the reaction is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, preferably 2 minutes to 1 hour.

In the step [1] of Aspects 6 to 10, the sample can be pretreated with an aqueous medium described later and then subjected to the reaction. Examples of the pretreatment include dilution, solubilization, denaturation and the like. The aqueous medium may contain metal ions, sugars, preservatives, proteins and the like, which will be described later. The temperature at which the sample is pretreated with an aqueous medium is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and 20 to 20 to. 40 ° C is particularly preferred. The time for pretreating the sample with an aqueous medium is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, preferably 2 minutes to 1 hour.

The method for measuring the labeling substance in the step [2] in the sixth aspect and the step [3] in the seventh to tenth aspects is not particularly limited as long as it is a method capable of measuring the steroid hormone of the present invention. The method and the like can be mentioned.

The steroid hormone in the present invention is not particularly limited as long as it is a steroid hormone capable of measuring the present invention, and examples thereof include mineralocorticoids, sugar corticoids, sex hormones, and the like, and mineralocorticoids are preferable. Examples of the mineralocorticoid include aldosterone and fludrocortisone acetate. Examples of the glucocorticoid include cortisol, corticosterone, cortisone and the like. Examples of sex hormones include male hormones (androgens) and female hormones. Examples of male hormones include testosterone, dehydroepiandrosterone and the like. Examples of female hormones include estradiol, estriol, estrone, progesterone and the like.

The sample in the present invention is not particularly limited as long as it is a sample capable of measuring the steroid hormone of the present invention, and examples thereof include whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, sweat, pancreatic fluid and the like. Therefore, whole blood, plasma, serum, urine and the like are preferable.

Examples of the buffer (I) in the present invention include 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES). , 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N'-(2-sulfoethyl) piperazin (HEPES), bis (2-) Hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) ), 3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2- Aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and the like, 2-morpholinoetansulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N'- (2-Sulfoethyl) piperazin (HEPES) and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) are preferred.

The concentration of the buffer (I) in the reaction solution of the reaction between the sample or the labeled competing substance and the antisteroid hormone antibody is not particularly limited as long as it is a concentration capable of measuring the steroid hormone of the present invention. Usually, it is 0.001 to 2.0 moL / L, preferably 0.005 to 1.0 moL / L.

The nonionic surfactant in the present invention is not particularly limited as long as it is a nonionic surfactant capable of measuring the steroid hormone of the present invention. For example, a polyoxyethylene-based nonionic surfactant or a non-ionic surfactant. Examples thereof include polyoxyethylene-based nonionic surfactants, and polyoxyethylene-based nonionic surfactants are preferable. Examples of the polyoxyethylene-based nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polyoxypropylene condensate, and polyoxyethylene alkyl amine. Examples thereof include alkylenediamine-polyoxyethylene polyoxypropylene condensate, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of the non-polyoxyethylene non-ionic surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, fatty acid alkanolamide, sucrose fatty acid ester and the like.

Examples of the polyoxyethylene alkyl ether include polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene dodecyl ether and the like.

Examples of the polyoxyethylene alkyl phenyl ether include polyoxyethylene octyl phenyl ether and polyoxyethylene nonyl phenyl ether.

Examples of the polyoxyethylene polycyclic phenyl ether include polyoxyethylene dibenzylphenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene (distyrene) phenyl ether, polyoxyethylene (tristyrene) phenyl ether and the like. Be done.

Examples of the polyoxyethylene polyoxypropylene condensate include a polyoxyethylene polyoxypropylene copolymer and a polyoxyethylene polyoxypropylene random polymer.

Examples of the polyoxyethylene alkylamine include polyoxyethylene dodecylamine and polyoxyethylene stearylamine.

Examples of the alkylenediamine-polyoxyethylene polyoxypropylene condensate include ethylenediamine tetrapolyoxyethylene, polyoxypropylene, and block polymer.

Examples of the polyoxyethylene fatty acid ester include polyoxyethylene glycol monolaurate, polyoxyethylene glycol monostearate, polyoxyethylene glycol distearate, and polyoxyethylene glycol monooleate.

Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan tristearate. Examples thereof include polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tetraoleate.

Examples of the sorbitan fatty acid ester include sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan distearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, and sorbitan sesquioleate.

Examples of the glycerin fatty acid ester include stearic acid monoglyceride and oleic acid monoglyceride.

Examples of the fatty acid alkanolamide include lauric acid diethanolamide and the like. Examples of the sucrose fatty acid ester include sucrose palmitic acid ester and sucrose stearic acid ester.

Examples of the anionic surfactant include carboxylates, sulfonates, sulfates, phosphates, oleic acids and the like.

Examples of the carboxylate include salts of higher fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid and alkali metals such as sodium and potassium. Specific examples thereof include potassium oleate, sodium lauroyl sarcosine, sodium N-myristoyl-N-methyl-β-alanine, sodium polyoxyethylene dodecyl ether acetate and the like.

Examples of the sulfonate include alkylbenzene sulfonic acids such as dodecylbenzene sulfonic acid, naphthalene sulfonic acids such as dipropylnaphthalene sulfonic acid and dibutylnaphthalene sulfonic acid, sulfosuccinic acids such as dioctyl sulfosuccinic acid, and salts with sodium and the like. Can be mentioned. Specific examples thereof include sodium dodecylbenzene sulfonate, sodium dipropylnaphthalene sulfonate, sodium dibutylnaphthalene sulfonate, sodium dioctyl sulfosuccinate and the like.

Examples of the sulfate ester salt include higher alcohol sulfate ester salt, polyoxyethylene alkyl ether sulfate ester salt, polyoxyethylene polycyclic phenyl ether sulfate ester salt and the like. Examples of the salt include sodium salt, ammonium salt and the like. Specific examples of the higher alcohol sulfate ester salt include sodium dodecyl sulfate and ammonium dodecyl sulfate, and specific examples of the polyoxyethylene alkyl ether sulfate ester salt include polyoxyethylene lauryl ether sulfate sodium and the like. Specific examples of the ring phenyl ether sulfate ester salt include polyoxyethylene polycyclic phenyl ether sulfate sodium and the like.

Examples of the phosphoric acid ester salt include a monoalkyl phosphate ester salt and a polyoxyethylene alkyl ether phosphoric acid ester salt. Examples of the salt include sodium salt, potassium salt and the like. Specific examples of the monoalkyl phosphate ester salt include sodium monostearyl phosphate and sodium monododecyl phosphate, and specific examples of the polyoxyethylene alkyl ether phosphate salt include polyoxyethylene dodecyl ether phosphate. Examples include potassium.

Examples of cholic acids include cholic acid, taurocholic acid, glycocholic acid, lithocolic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, 7-oxolithocolic acid, 12-oxolithocolic acid, 12-oxokenodeoxycholic acid, 7 -Oxodeoxycholic acid, hyocholic acid, hyodeoxycholic acid, dehydrocholic acid and the like can be mentioned. These cole acids may form salts. Examples of the salt include ammonium salt, lithium salt, sodium salt, potassium salt, magnesium salt, calcium salt and the like.

Examples of amphoteric surfactants include betaines, glycines, alanines, 2-alkylimidazoline derivatives, amine oxides, alkylamino salts, and bile acids.

Examples of betaines include alkyl betaines, carboxybetaines, sulfobetaines and the like. Examples of alkyl betaines include N-dodecyl betaine and the like. Examples of the carboxybetaines include amidopropyl betaine laurate, betaine dodecyldimethylaminoacetate, N-lauroyl-N'-carboxymethyl-N'-hydroxyethylethylenediamine sodium and the like. Examples of sulfobetaines include amide propyl hydroxysulfobetaine laurate.

Examples of glycines include sodium dodecyldiaminoethylglycine and the like.

Examples of alanines include N-octodesyl-β-alanine sodium, N-mystylyl-β-alanine sodium, sodium laurylaminopropionate and the like.
Examples of the 2-alkylimidazoline derivative include 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine such as 2-lauroyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine.

Examples of amine oxides include dodecyldimethylamine oxide, dihydroxyethyl dodecylamine oxide, polyoxyethylene coconut oil alkyldimethylamine oxide and the like.

Examples of alkylamino salts include sodium dodecylaminodiacetate and the like.

Examples of bile acids include 3-[(3-colamidepropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-colamidepropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), and the like. Can be mentioned.

The concentration of the nonionic surfactant, the anionic surfactant, or the amphoteric surfactant in the reaction solution of the reaction between the sample or the labeling competitor and the antisteroid hormone antibody is the steroid hormone of the present invention. The concentration is not particularly limited as long as it enables the measurement of, and is usually 0.005 to 3%, preferably 0.01 to 2%. The concentration of the nonionic surfactant, the anionic surfactant, or the amphoteric surfactant in the reaction solution of the reaction between the sample or the competing substance and the labeled antisteroid hormone antibody is the concentration of the present invention. The concentration is not particularly limited as long as it enables measurement of steroid hormones, and is usually 0.005 to 3%, preferably 0.01 to 2%.

In the measuring method of the present invention, the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant may be used alone or in combination of two or more.

The insoluble carrier in the present invention is not particularly limited as long as it is an insoluble carrier that immobilizes an antisteroid hormone antibody and a competing substance and enables measurement of the steroid hormone of the present invention. For example, a polystyrene plate such as a microtiter plate or glass Examples thereof include granules (beads) made of glass or synthetic resin, spheres (balls) made of glass or synthetic resin, various membranes such as latex, magnetic particles, nitrocellulose membrane, and test tubes made of synthetic resin.

In the present invention, the bond between the antisteroid hormone antibody and the insoluble carrier is not particularly limited as long as it is a bond capable of measuring the steroid hormone of the present invention, and examples thereof include physical adsorption and chemical bond. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordination bond.

The anti-steroid hormone antibody may be directly immobilized on the insoluble carrier or indirectly immobilized on the insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. Examples of the indirect immobilization method include a method of immobilizing an antisteroid hormone antibody on an insoluble carrier via a specific binding between biotin and avidins (avidin, streptavidin, neutravidin, etc.). In addition, the anti-steroid hormone antibody may be immobilized on an insoluble carrier by covalent bonding via a linker.

Examples of the linker include molecules capable of covalently binding both the functional group on the surface of the insoluble carrier and the functional group possessed by the antisteroid hormone antibody, and the first reaction activity capable of reacting with the functional group possessed by the antisteroid hormone antibody. A molecule having a group and a second reactive active group capable of reacting with a functional group on the surface of an insoluble carrier in the same molecule, and the first reactive active group and the second reactive active group are different groups. preferable. Examples of the functional group retained on the surface of the functional group of the antisteroid hormone antibody and the insoluble carrier include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, and an N-hydroxysuccinyl group ( NHS group), maleimide group and the like. Examples of reactive groups in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, solarene, pyridyl disulfide, and the like. Examples include groups such as vinyl sulfone.

In the present invention, the bond between the competing substance and the insoluble carrier is not particularly limited as long as it is a bond capable of measuring the steroid hormone of the present invention, and examples thereof include physical adsorption and chemical bond. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordination bond.

The competing substance may be directly immobilized on the insoluble carrier or indirectly immobilized on the insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. Examples of the indirect immobilization method include a method of immobilizing an antisteroid hormone antibody on an insoluble carrier via a specific binding between biotin and avidins (avidin, streptavidin, neutravidin, etc.). In addition, the competing substance may be immobilized on the insoluble carrier by covalent bonding via a linker.

Examples of the linker include molecules capable of covalently bonding both the functional group on the surface of the insoluble carrier and the functional group possessed by the competing substance, and the first reactive group capable of reacting with the functional group possessed by the competing substance and the insoluble group. A molecule having a second reactive active group capable of reacting with a functional group on the surface of the carrier in the same molecule, and the first reactive active group and the second reactive active group are different groups is preferable. Examples of the functional group of the competing substance and the functional group retained on the surface of the insoluble carrier include the above-mentioned functional group and the like. Examples of the reactive group in the linker include the above-mentioned reactive group.

The anti-steroid hormone antibody in the present invention is not particularly limited as long as it is an antibody that binds to the steroid hormone and enables the measurement of the steroid hormone of the present invention, and either a polyclonal antibody or a monoclonal antibody can be used. Further, in the present invention, not only a full-length antibody but also an antibody fragment can be used. Examples of the antibody fragment include an antibody fragment obtained by removing the Fc portion such as Fab obtained by papain treatment of the antibody, F (ab') ₂ obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment. Can be mentioned. When an insoluble carrier on which avidins (avidin, neutravidin, streptavidin, etc.) are immobilized is used as the insoluble carrier, a biotin-binding antisteroid hormone antibody in which biotin is bound to the antisteroid hormone antibody should be used. Can be done. When an insoluble carrier on which biotin is immobilized is used as the insoluble carrier, an avidin-bound antisteroid hormone antibody in which avidins (avidin, neutravidin, streptavidin, etc.) are bound to the antisteroid hormone antibody. Can be used.

The anti-steroid hormone antibody in the present invention can be produced by a usual method for producing an antibody using the steroid hormone itself or a compound having a partial structure corresponding to an epitope in the steroid hormone as an antigen, but it can also be produced as a commercially available product. It is available. Examples of commercially available anti-steroid hormone antibodies include anti-steroid hormone polyclonal antibodies (manufactured by Abcam).

The labeled anti-steroid hormone antibody in the present invention is produced by the method described below using the above-mentioned anti-steroid hormone antibody and the labeling substance described below.

The competing substance in the present invention means a substance that binds to an antisteroid hormone antibody and the binding thereof is competitive with the steroid hormone that is the measurement target substance, and also includes the steroid hormone itself that is the measurement target substance. .. As the competing substance in the present invention, a substance having the same structure as the epitope structure recognized by the antisteroid hormone antibody is preferable, and the strength of binding to the antisteroid hormone antibody is such that the binding of the steroid hormone to the antibody is strong. It is more preferable that the strength is the same as that of steroid hormone itself, and specific examples thereof include steroid hormone itself, which is a substance to be measured.

When an insoluble carrier on which avidins (avidin, neutravidin, streptavidin, etc.) are immobilized is used as the insoluble carrier, a biotin-binding competing substance in which biotin is bound to the competing substance can be used. When an insoluble carrier on which biotin is immobilized is used as the insoluble carrier, an avidin-binding competing substance in which avidins (avidin, neutravidin, streptavidin, etc.) are bound to a competing substance may be used. can.

The competing substance in the present invention can be produced by a known method, for example, by the methods described in JP-A-60-260592 and JP-A-63-060996.

The labeling competing substance in the present invention is produced by the method described below using the above-mentioned competing substance and the labeling substance described below.

The labeling substance used in the labeled antisteroid hormone antibody and the labeling competing substance in the present invention is not particularly limited as long as it is a labeling substance capable of measuring the steroid hormone of the present invention, for example, an enzyme, a fluorescent substance, and the like. Examples thereof include luminescent substances, radioactive isotopes, biotin, digoxigenin, polypeptides containing tag sequences, metal colloid particles, colored latex particles and the like.

Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.

Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine B-isothiocyanate (RITC). Other fluorescent substances include, for example, quantum dot (Science, 281, 2016-2018, 1998), phycobilly protein such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein). , BFP (Blue fluorescent Protein) and other proteins that emit fluorescence.

Examples of the luminescent substance include aclidinium and its derivatives, ruthenium complex compounds, loffin and the like. As the ruthenium complex compound, those shown in Clin. Chem. 37, 9, 1534-1539, 1991, which emit light electrochemically together with an electron donor, are preferable.
Examples of the radioactive isotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, ¹³¹ I and the like.

Examples of the polypeptide containing the tag sequence include FLAG peptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Lys), polyhistidine (His tag, His His His His His), and myc epitope peptide (myc tag, Glu Gln Lys Leu). Ile Ser Glu Glu Glu Asp Leu), hemagglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) and the like.

Labeling of the antisteroid hormone antibody with the labeling substance and labeling of the competing substance with the labeling substance are carried out via a linker between the functional group of the antisteroid hormone antibody or the competing substance and the functional group of the labeling substance. Alternatively, it can be carried out by a reaction that causes a covalent bond without intervention. Examples of the functional group include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, a hydroxysuccinyl ester group, a maleimide group, an isothiocyanate group and the like.

In labeling a competing substance with a labeling substance, the position in the competing substance at which the competing substance binds to the labeling substance can be appropriately selected depending on the structure of the competing substance. For example, when the steroid hormone to be measured is aldosterone and aldosterone itself is used as a competing substance, the labeling substance can be bound to positions such as the 3-position, 4-position, 6-position or 21-position of aldosterone.

Examples of the linking method without a linker include a method using a carbodiimide compound such as 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDC). In this case, it is also possible to use an active ester such as NHS or a derivative thereof. The condensation reaction between the isothiocyanate group and the amino group is preferable because it does not require any other reagent and proceeds only by mixing under neutral to weakly alkaline conditions.

The linker may be any molecule as long as it can bind the functional group of the anti-steroid hormone antibody or competing substance to the functional group of the labeling substance. In a preferred embodiment, for example, a first functional group capable of reacting with an amino acid residue of an antisteroid hormone antibody and a second functional group capable of reacting with a functional group of a labeling substance are contained in the same molecule. Examples thereof include a molecule having a first functional group capable of reacting with a carboxyl group of a competing substance and a molecule having a second functional group capable of reacting with a functional group of a labeling substance in the same molecule. It is preferable that the first functional group and the second functional group are different. Examples of the functional group of the linker include the above-mentioned functional groups.

Examples of the method for chemically bonding radioisotopes include the methods described in the literature (Antibody Immunoconj. Radiopharm., 3, 60, 1990).

When the labeling substance is a polypeptide such as an enzyme, avidin, a fluorescent protein, a phycobili protein, or a polypeptide containing a tag sequence, a known gene recombination technique (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring) According to Harbor Laboratory Press, 2001), an expression vector containing a DNA encoding a fusion protein of a labeling substance and an antisteroid hormone antibody was prepared, the expression vector was introduced into an appropriate host, and the host was cultured. can do. The DNA encoding the fusion protein can be obtained by cloning the DNA encoding the antibody and the labeling substance by PCR or the like and ligating the respective DNAs by a ligase reaction.

In the present invention, the measurement of the labeling substance in the immune complex of the anti-steroid hormone antibody and the labeling competitor and the measurement of the labeling substance in the immune complex of the labeled anti-steroid hormone antibody and the competition substance are used. It can be appropriately selected depending on the labeling substance.

When the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength, the absorbance can be measured using a spectrophotometer, a multi-well plate reader, or the like.
When the labeling substance is a fluorescent substance, the fluorescence intensity can be measured using a fluorometer, a fluorescent multi-well plate reader, or the like.
When the labeling substance is a luminescent substance, the luminescence intensity can be measured using a luminescent photometer, a luminescent multi-well plate reader, or the like.
When the labeling substance is a radioisotope, the radioactivity can be measured by a scintillation counter, a γ-well counter, or the like.

When the labeling substance is an enzyme, the labeling amount can be quantified by measuring the enzyme activity. For example, the labeled amount can be measured by reacting the substrate of an enzyme with the enzyme and measuring the produced substance.

When the enzyme is peroxidase, the peroxidase activity can be measured by, for example, an absorptiometry or a fluorescence method. As a method for measuring peroxidase activity by the absorbance method, for example, peroxidase is reacted with a combination of hydrogen peroxide, which is a substrate thereof, and an oxidative color-developing chromogen, and the absorbance of the reaction solution is measured by a spectrophotometer or a multi-well plate reader. And the like. Examples of the oxidative color-developing chromogen include a leuco-type chromogen, an oxidative-coupling chromogen, and the like.

The leuco-type chromogen is a substance that is independently converted into a dye in the presence of an active peroxide substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 10-N-carboxymethylcarbamoyl-3 , 7-Bis (dimethylamino) -10H-Phenothiazine sodium salt (DA-67), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] Amine (BCMA) and the like can be mentioned.

Oxidation-coupling color-developing chromogen is a substance in which two compounds oxidatively couple to form a dye in the presence of peroxidase-active substances such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and anilines (Trinder's reagent), a combination of a coupler and phenols, and the like.

Examples of the coupler include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazine and the like.

Examples of anilins include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), and N-ethyl-N- (2-hydroxy). -3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-Bis (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-Ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3 , 5-Dimethylaniline, N-Ethyl-N- (2-Hydroxy-3-sulfopropyl) -3-methoxyaniline, N-Ethyl-N-(2-Hydroxy-3-sulfopropyl) aniline, N-Ethyl- N- (3-Methylphenyl) -N'-Succinylethylene Diamine (EMSE), N-Ethyl-N- (3-Methylphenyl) -N'-Acetylethylene Diamine, N-Ethyl-N- (2-Hydroxy-3-) Sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS) and the like can be mentioned.

Examples of phenols include phenol, 4-4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

As a method for measuring peroxidase activity by the fluorescence method, for example, peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as its substrate, and the intensity of fluorescence generated by a fluorometer, a fluorescent multi-well plate reader, or the like is measured. Examples include a measuring method. Examples of the fluorescent substance include 4-hydroxyphenyl acetic acid, 3- (4-hydroxyphenyl) propionic acid, coumarin and the like.

As a method for measuring peroxidase activity by the luminescence method, for example, peroxidase is reacted with a combination of hydrogen peroxide as a substrate and a luminescent substance, and the intensity of luminescence generated by a luminescence intensity meter, a luminescent multi-well plate reader, or the like is measured. Examples include a measuring method. Examples of the luminescent substance include a luminol compound and a lucigenin compound.

When the enzyme is alkaline phosphatase, the alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring the alkaline phosphatase activity by the luminescence method include a method in which alkaline phosphatase is reacted with its substrate and the luminescence intensity of the generated luminescence is measured with a luminescence intensity meter, a luminescence multi-well plate reader, or the like.

Examples of the substrate for alkaline phosphatase include 3- (2'-spirodamantan) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane / disodium salt (AMPPD), 2-chloro-. 5- {4-Methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo [3.3.1.13.7] decane] -4-yl} phenyl phosphate disodium salt (CDP-Star) ^TM ), 3- {4-Methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo [3.3.1.13.7] decane] 3.3.1.13.7 yl} phenyl phosphate disodium Examples thereof include salt (CSPD ^TM ), [10-methyl-9 (10H) -acridinylidene] phenoxymethylphosphate / disodium salt (Lumigen ^TM APS-5), and the like.

When the enzyme is β-D-galactosidase, the β-D-galactosidase activity can be measured by, for example, an absorptiometry (colorimetric method), a luminescence method, a fluorescence method, or the like. As a method for measuring β-D-galactosidase activity by the absorbance method (colorimetric method), for example, β-D-galactosidase is reacted with its substrate, and the absorbance of the reaction solution is measured with a spectrophotometer, a multi-well plate reader, or the like. Examples include a measuring method. Examples of the substrate for β-D-galactosidase include o-nitrofer-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by the luminescence method, for example, β-D-galactosidase is reacted with its substrate, and the luminescence of the reaction solution is measured with a luminescence intensity meter, a luminescence multi-well plate reader, or the like. And so on. Examples of the substrate for β-D-galactosidase include Galacton-Plus [manufactured by Applied Biosystems] and similar compounds thereof. As a method for measuring β-D-galactosidase activity by the fluorescence method, for example, β-D-galactosidase is reacted with its substrate, and the fluorescence of the reaction solution is measured with a fluorometer, a fluorescence multi-well plate reader, or the like. And so on. Examples of the substrate for β-D-galactosidase include 4-methylumbelliferyl-β-D-galactopyranoside.

When the enzyme is luciferase, the luciferase activity can be measured by, for example, a luminescence method. Examples of the method for measuring the luciferase activity by the luminescence method include a method in which luciferase is reacted with its substrate and the luminescence of the reaction solution is measured with a luminescence intensity meter, a luminescence multi-well plate reader, or the like. Examples of the substrate for luciferase include luciferin and coelenterazine.

When the labeling substance is other than a fluorescent substance, a luminescent substance, a radioactive isotope, and an enzyme, the substance that specifically binds to the labeling substance is labeled with a fluorescent substance, a luminescent substance, a radioactive isotope, an enzyme, or the like, and immunity. Fluorescent substances, luminescent substances, and radioactive substances that bind to the labeling substance constituting the labeled antisteroid hormone antibody or labeling competing substance in the complex and label the substance that specifically binds to the labeling substance. The labeling substance can be measured by measuring the isotope or enzyme by the method described above. Examples of the substance that specifically binds to the labeling substance include an antibody that specifically binds to the labeling substance, and avidins and the like when the labeling substance is biotin. In addition, a substance that specifically binds to the labeling substance (antibodies that specifically bind to the labeling substance, avidins, etc.) is used to bind to the labeling substance in the immune complex, and then specifically bind to the labeling substance. Antibodies that specifically bind to substances that bind to substances, such as antibodies that specifically bind to the constant region of antibodies that specifically bind to labeling substances, or antibodies that specifically bind to avidins, such as fluorescent substances, luminescent substances, and radioactive isotopes. Alternatively, the labeled substance can be measured by binding an enzyme-labeled substance and measuring a fluorescent substance, a luminescent substance, a radioactive isotope or an enzyme labeling these antibodies by the above-mentioned method.

The aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium capable of measuring the steroid hormone of the present invention, and examples thereof include deionized water and distilled water.

Examples of the metal ion in the present invention include magnesium ion, manganese ion, zinc ion and the like. Examples of the saccharide in the present invention include mannitol, sorbitol and the like. Examples of the preservative in the present invention include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), bioace, Proclin 300, Proxel GXL, and the like. Examples of the protein in the present invention include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, block ace (manufactured by DS Pharma Biomedical) and the like.

(Reagent for measurement)
The steroid hormone measuring reagent in the sample of the present invention is a reagent used in the steroid hormone measuring method of the present invention, and is composed of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. It is a reagent containing at least one surfactant selected from the above group, a buffer (I), a labeling competitor, and an antisteroid hormone antibody. The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but it is preferable that it is immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

Further, the reagent for measuring the steroid hormone in the sample of the present invention is at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. A reagent comprising a buffer (I), a labeled anti-steroid hormone antibody, and a competing substance. The competing material may or may not be immobilized on the insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

The measurement reagent of the present invention may be in a freeze-dried state or in a liquid state. When a reagent for measurement in a freeze-dried state is used, it is dissolved in an aqueous medium to make it liquid and used for measurement before measurement. In the liquid measurement reagent, at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, a buffer (I), and labeling. Competitive substances, labeled anti-steroid hormone antibodies, competing substances, and anti-steroid hormone antibodies are in a state of being dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like. Liquid measurement reagents include aqueous media. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like.

Examples of the steroid hormone in the measurement reagent of the present invention include the above-mentioned steroid hormones. Examples of the buffer (I) in the measurement reagent of the present invention include the above-mentioned buffer (I). The content of the buffer (I) in the measurement reagent of the present invention is not particularly limited as long as it is a content capable of measuring the steroid hormone of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned aqueous medium. The content is usually 0.001 to 2.0 moL / L in the state of being dissolved in the medium, and the content is preferably 0.005 to 1.0 moL / L.

Examples of the nonionic surfactant, anionic surfactant, and amphoteric surfactant in the measuring reagent of the present invention include the above-mentioned nonionic surfactant, anionic surfactant, and amphoteric. Surfactants can be mentioned respectively. The content of each of the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant in the measuring reagent of the present invention is a content that enables the measurement of the steroid hormone of the present invention. If this is the case, there is no particular limitation, and the content is usually 0.005 to 3% in the above-mentioned aqueous medium or in a state of being dissolved in the above-mentioned aqueous medium, and a content of 0.01 to 2% is preferable.

In the measurement reagent of the present invention, the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant may be used alone or in combination of two or more.

Labeled competing substances, anti-steroid hormone antibodies, labeled anti-steroid hormone antibodies, and competing substances in the measurement reagents of the present invention include, for example, the above-mentioned labeled competing substances, anti-steroid hormone antibodies, labeled anti-steroid hormone antibodies, and competing substances. Each substance is listed.

In the measurement reagent of the present invention, instead of the insoluble carrier on which the anti-steroid hormone antibody is immobilized, the anti-steroid hormone antibody to which one of the set of affinity substances (A) is bound and the set of affinity substances are used. A combination with an insoluble carrier to which the other (a) is bound can also be used. In this case, the insoluble carrier on which the anti-steroid hormone antibody is immobilized is produced in the reaction solution of the reaction between the sample or the labeled competing substance and the anti-steroid hormone antibody. Examples of the combination of a set of affinity substances, that is, the combination of A and a include the above-mentioned combinations and the like.

Further, in the measurement reagent of the present invention, instead of the insoluble carrier on which the competing substance is immobilized, the competing substance to which one of the set of affinity substances (B) is bound and the other of the set of affinity substances are bound. A combination with an insoluble carrier to which (b) is bound can also be used. In this case, the insoluble carrier on which the competing substance is immobilized is produced in the reaction solution of the reaction between the sample or the competing substance and the labeled anti-steroid hormone antibody. Examples of the combination of a set of affinity substances, that is, the combination of B and b include the above-mentioned combinations and the like.

The measurement reagent of the present invention may include a sample pretreatment reagent for pretreating a sample. Examples of the pretreatment include dilution, solubilization, denaturation and the like. Examples of the sample pretreatment reagent include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like. The sample pretreatment reagent may be in a liquid state or in a lyophilized state. When a freeze-dried sample pretreatment reagent is used, it can be used for sample pretreatment after being previously dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like.

(Measurement kit)
The measurement reagent of the present invention can also take the form of a kit from the viewpoint of storage, transportation, distribution and the like. The measurement kit of the present invention is used in the method for measuring a steroid hormone of the present invention.

The kit for measuring steroid hormones in the sample of the present invention contains a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeling competitor, and contains a nonionic surfactant and an anionic surfactant. A kit in which at least one surfactant selected from the group consisting of an activator and an amphoteric surfactant and a buffer (I) are contained in either or both of the first reagent and the second reagent. be.

In the measurement kit of the present invention, the anti-steroid hormone antibody may or may not be immobilized on the insoluble carrier, but it is preferable that it is immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

In addition, the kit for measuring steroid hormones in the sample of the present invention contains a first reagent containing a labeled antisteroid hormone antibody and a second reagent containing a competing substance, and is a nonionic surfactant and anion. At least one surfactant selected from the group consisting of a sex surfactant and an amphoteric surfactant, and each of the buffer (I) are contained in either or both of the first reagent and the second reagent. It is a kit.

In the measurement kit of the present invention, the competing substance may or may not be immobilized on the insoluble carrier, but it is preferably immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

The measurement kit of the present invention may be in a lyophilized state or in a liquid state. When a freeze-dried measurement kit is used, it is dissolved in an aqueous medium to make it liquid and used for measurement before measurement. In the liquid measurement kit, at least one surfactant, buffer (I), and labeling selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants. Competitive substances, labeled anti-steroid hormone antibodies, competing substances, and anti-steroid hormone antibodies are in a state of being dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like. The liquid measurement kit contains an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like.

Examples of the steroid hormone in the measurement kit of the present invention include the above-mentioned steroid hormones. Examples of the buffer (I) in the measurement kit of the present invention include the above-mentioned buffer (I). As the content of the buffer (I) in the first reagent containing the antisteroid hormone antibody of the measurement kit of the present invention and the second reagent containing the labeling competing substance, the steroid hormone of the present invention can be measured. The content is not particularly limited, and is usually 0.001 to 2.0 moL / L in the above-mentioned aqueous medium or in a state of being dissolved in the aqueous medium, and 0.005 to 1.0 moL / L. Is preferable. The content of the buffer (I) in the first reagent containing the labeled antisteroid hormone antibody of the measurement kit of the present invention and the second reagent containing a competing substance is the measurement of the steroid hormone of the present invention. The content is not particularly limited as long as it enables the above-mentioned content, and is usually 0.001 to 2.0 moL / L in the above-mentioned aqueous medium or in a state of being dissolved in the aqueous medium, and 0.005 to 1.0 moL / L. Content is preferred.

Examples of the nonionic surfactant, anionic surfactant, and amphoteric surfactant in the measurement kit of the present invention include the above-mentioned nonionic surfactant, anionic surfactant, and amphoteric. Surfactants can be mentioned respectively. Nonionic surfactants, anionic surfactants, and amphoteric surfactants in the first reagent containing the antisteroid hormone antibody of the measurement kit of the present invention and the second reagent containing a labeling competitor. The content of each surfactant of the agent is not particularly limited as long as it is a content capable of measuring the steroid hormone of the present invention, and is usually in the above-mentioned aqueous medium or in a state of being dissolved in the aqueous medium. The content is 0.02 to 10%, preferably 0.04 to 5%. In addition, a nonionic surfactant, an anionic surfactant, and amphoteric in the first reagent containing the labeled antisteroid hormone antibody of the measurement kit of the present invention and the second reagent containing a competing substance. The content of each surfactant of the surfactant is not particularly limited as long as it is a content that enables the measurement of the steroid hormone of the present invention, and is usually dissolved in the above-mentioned aqueous medium or in the aqueous medium. The content is 0.02 to 10% in the state, and the content is preferably 0.04 to 5%.

In the measurement kit of the present invention, the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant may be used alone or in combination of two or more.

Labeled competing substances, anti-steroid hormone antibodies, labeled anti-steroid hormone antibodies, and competing substances in the measurement kit of the present invention include, for example, the above-mentioned labeled competing substances, anti-steroid hormone antibodies, labeled anti-steroid hormone antibodies, and competing substances. Each substance is listed.

In the measurement kit of the present invention, instead of an insoluble carrier on which an anti-steroid hormone antibody is immobilized, an anti-steroid hormone antibody to which one of a set of affinity substances (A) is bound and a set of affinity substances are used. A combination with an insoluble carrier to which the other (a) is bound can also be used. In this case, the insoluble carrier on which the anti-steroid hormone antibody is immobilized is produced in the reaction solution of the reaction between the sample or the labeled competing substance and the anti-steroid hormone antibody. In the measurement kit of the present invention, a combination of an anti-steroid hormone antibody to which one of a set of affinity substances (A) is bound and an insoluble carrier to which the other (a) of a set of affinity substances is bound is used. In the case, even if the anti-steroid hormone antibody to which one of the set of affinity substances (A) is bound and the insoluble carrier to which the other (a) of the set of affinity substances is bound are contained in the same reagent, It may be contained in separate reagents, but is preferably contained in separate reagents. A kit in which an anti-steroid hormone antibody to which one of a set of affinity substances (A) is bound and an insoluble carrier to which the other (a) of a set of affinity substances is bound are contained in separate reagents. In the case, a first reagent containing an anti-steroid hormone antibody to which one of the set of affinity substances (A) is bound, a second reagent containing a labeling competitor, and the other (a) of the set of affinity substances. A kit containing a third reagent containing an insoluble carrier to which the antibody is bound is also included in the kit of the present invention. Examples of the combination of a set of affinity substances, that is, the combination of A and a include the above-mentioned combinations and the like. When an insoluble carrier on which an anti-steroid hormone antibody is immobilized is produced in a reaction solution for an antigen-antibody reaction, examples of the measurement kit of the present invention include the following kits.
・ Kit 1
A first reagent containing an antisteroid hormone antibody to which one of a set of affinity substances (A) is bound, a labeling competitor, and an insoluble carrier to which the other (a) of a set of affinity substances is bound. A second reagent containing, at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, and a buffer (I). A kit in which each is contained in one or both of the first reagent and the second reagent.
・ Kit 2
A first reagent containing an antisteroid hormone antibody to which one of a set of affinity substances (A) is bound, a second reagent containing a labeling competitor, and the other (a) of a set of affinity substances are bound. At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, which contains a third reagent containing the insoluble carrier, and a buffering agent. A kit in which each of (I) is contained in at least one reagent of the first reagent, the second reagent, and the third reagent.

In the measurement kit of the present invention, instead of the insoluble carrier on which the competing substance is immobilized, the competing substance to which one of the set of affinity substances (B) is bound and the other of the set of affinity substances (b). ) Can also be used in combination with an insoluble carrier bound. In this case, the insoluble carrier on which the competing substance is immobilized is produced in the reaction solution of the reaction between the sample or the competing substance and the labeled anti-steroid hormone antibody. In the measurement kit of the present invention, when a combination of a competing substance to which one of a set of affinity substances (B) is bound and an insoluble carrier to which the other (b) of a set of affinity substances is bound is used. Even if the competing substance to which one of the set of affinity substances (B) is bound and the insoluble carrier to which the other (b) of the set of affinity substances is bound are contained in the same reagent, they are contained in different reagents. It may be included, but is preferably contained in separate reagents. In the case of a kit in which a competing substance to which one of a set of affinity substances (B) is bound and an insoluble carrier to which the other (b) of a set of affinity substances is bound are contained in separate reagents. A first reagent containing a labeled anti-steroid hormone antibody, a second reagent containing a competing substance to which one of the set of affinity substances (B) is bound, and the other (b) of the set of affinity substances are bound. A kit containing a third reagent containing the insoluble carrier is also included in the kit of the present invention. Examples of the combination of a set of affinity substances, that is, the combination of B and b include the above-mentioned combinations and the like. When an insoluble carrier on which a competing substance is immobilized is produced in a reaction solution for an antigen-antibody reaction, examples of the measurement kit of the present invention include the following kits.
・ Kit 3
A first reagent containing a labeled anti-steroid hormone antibody and an insoluble carrier to which the other (b) of the set of affinity substances is bound, and a competing substance to which one of the sets of affinity substances (B) is bound. A second reagent containing, at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, and a buffer (I). A kit in which each is contained in one or both of the first reagent and the second reagent.
・ Kit 4
A first reagent containing a labeled antisteroid hormone antibody, a second reagent containing a competing substance to which one of the set of affinity substances (B) is bound, and the other (b) of the set of affinity substances are bound. At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, which contains a third reagent containing the insoluble carrier, and a buffering agent. A kit in which each of (I) is contained in at least one reagent of the first reagent, the second reagent, and the third reagent.

In the measurement kit of the present invention, a sample pretreatment reagent for pretreating a sample may be included. Examples of the pretreatment include dilution, solubilization, denaturation and the like. Examples of the sample pretreatment reagent include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like. The sample pretreatment reagent may be in a liquid state or in a lyophilized state. When a freeze-dried sample pretreatment reagent is used, it can be used for sample pretreatment after being previously dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention at all.

[Reference example] Preparation of anti-aldosterone antibody (1) Preparation of immunogen
In a DMSO solution (25 μL) of 100 mg / mL ALDOSTERONE 3-CMO prepared by dissolving ALDOSTERONE 3-CMO (ALDOSTERONE 3-carbodimethyloxime; manufactured by SERALOIDS) with dimethylsulfoxide (DMSO) (manufactured by Sigma Aldrich). , 10 mg / mL KLH (Keyhole limpet hemocyanin; Thermo Fisher Scientific) PBS solution (2 mL), 100 mg / mL EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; Thermo-Fisher Scientific] DMSO solution (50 μL) and 100 mg / mL NHS (N-hydroxysuccinimide; Thermo-Fisher Scientific) DMSO solution (10 μL) were added and mixed, and 4 The reaction was carried out at ° C. for 16 hours to combine ALDOSTERONE 3-CMO and KLH to prepare a KLH-bound ALDOSTERONE 3-CMO solution. The prepared KLH-bound ALDOSTERONE 3-CMO solution was replaced with a solvent using a PD-10 column (manufactured by GE Healthcare Japan) equilibrated with the PBS solution to prepare a PBS solution of KLH-linked ALDOSTERONE 3-CMO. bottom. The obtained PBS solution of KLH-binding ALDOSTERONE 3-CMO was used as an immunogen.

(2) Preparation of anti-aldosterone polyclonal antibody The immunogen containing 0.5 mg of KLH-binding ALDOSTERONE 3-CMO prepared in (1) above was mixed with an adjuvant and emulsionized, and the obtained KLH-binding ALDOSTERONE 3-CMO emulsion was measured. It was subcutaneously injected into rabbits (NZW species) four times. Freund's Complete Adjuvant (manufactured by Sigma Aldrich) was used as an adjuvant for the first immunization, and Freund's Incomplete Adjuvant (manufactured by Sigma Aldrich) was used as an adjuvant for the second to fourth immunizations at 2-week intervals. After a total of 4 immunizations, blood was collected and the antibody titer in the serum was evaluated by ELISA. After confirming that the antibody titer in serum was elevated, Freund's Incomplete Adjuvant (manufactured by Sigma-Aldrich) was used as an adjuvant, and immunization was performed twice at 2-week intervals. Blood was collected after the final immunization, and this antiserum was purified using Protein A Sepharose (manufactured by GE Healthcare Japan) to obtain an anti-aldosterone polyclonal antibody.

Kits A, B, C, D and E for measuring aldosterone consisting of the following streptavidin-bound magnetic particle suspension, biotin-bound anti-aldosterone antibody solution, alkaline phosphatase-labeled competitor solution, and aldosterone standard solution were prepared.

<Streptavidin-bonded magnetic particle suspension>
As the magnetic particles, commercially available magnetic particles (Dynabeads M-280 Streptavidin: manufactured by Thermo Fisher Scientific Co., Ltd.) to which streptavidin was bound were used to prepare a magnetic particle suspension having the following composition.

Buffer (I) 125 mmol / L (see Tables 1 and 2)
Streptavidin-bonded magnetic particles 0.7 mg / mL
BSA 0.1%
Tween 20 1.0%

<Biotin-bound anti-aldosterone antibody solution>
Using the anti-aldosterone polyclonal antibody obtained in the reference example, the antibody and NHS-biotin (manufactured by Thermo Fisher Scientific) were mixed and reacted at 37 ° C. for 1 hour, and the mixture after the reaction was prepared as Sephadex G. The unreacted NHS-biotin was removed by subjecting to a -50 column (manufactured by GE Healthcare Japan Co., Ltd.) to prepare a biotin-binding anti-aldosterone polyclonal antibody. Using the obtained biotin-binding anti-aldosterone polyclonal antibody, a biotin-binding anti-aldosterone antibody solution having the following composition was prepared.

Buffer (I) 125 mmol / L (see Tables 1 and 2)
Biotin-binding anti-aldosterone antibody 22.5 ng / mL
BSA 1.0%

<Alkaline phosphatase-labeled competing substance solution>
ALDOSTERONE 3-CMO (manufactured by STERALOIDS) in a mixed solvent of DMSO-water (3: 1), Sulfo-NHS (manufactured by Thermo Fisher Scientific) and EDC [1-ethyl-3- (3-dimethyl). Aminopropyl) carbodiimide hydrochloride; Thermo Fisher Scientific], reacted at 25 ° C for 15 minutes, and then with alkaline phosphatase (Roche Diagnostics) for 16 hours at 4 ° C. It was reacted. The reaction mixture was subjected to a Sephadex G-50 column (manufactured by GE Healthcare Japan) to remove unreacted ALDOSTERONE 3-CMO to obtain an alkaline phosphatase-labeled competitor. Using the obtained alkaline phosphatase-labeled competing substance, an alkaline phosphatase-labeled competing substance solution having the following composition was prepared.

Buffer (I) 20 mmol / L (see Tables 1 and 2)
Alkaline phosphatase-labeled competitor 1.5 μg / mL
BSA 0.1%
Tween 20 1.0%
Magnesium chloride hexahydrate 30 mmol / L

<Aldosterone standard solution>
Aldosterone (manufactured by Sigma Aldrich) diluted with defatted plasma [Human Plasma, Defibrinated, Delipidized double charcoal stripped (manufactured by Golden West Biologicals)] with aldosterone concentration of 0, 16, 40, 80, Aldosterone solutions at 160, 400, and 1600 pg / mL concentrations were used as aldosterone standard solutions for plasma line preparation.

The same type of buffer was used for the streptavidin-bound magnetic particle suspension, the biotin-bound anti-aldosterone antibody solution, and the alkaline phosphatase-labeled competing substance solution.

[Comparative Example 1]
Using the same method except that a phosphate buffer solution, a Tris buffer solution or a citrate buffer solution is used instead of the buffer solution (I) of Example 1, a streptavidin-bound magnetic particle suspension, a biotin-bound anti-aldosterone is used. An antibody solution and an alkali phosphatase-labeled competing substance solution are prepared, and a kit consisting of each streptavidin-binding magnetic particle suspension, a biotin-binding anti-aldosterone antibody solution, an alkali phosphatase-labeled competing substance solution, and the aldosterone standard solution of Example 1 is prepared. , B and c were prepared.

(1) Preparation of calibration line To the reaction vessel, each aldosterone standard solution (10 μL) of Example 1 and the biotin-binding anti-aldosterone antibody solution (50 μL) of Example 1 were added and stirred, and the mixture was stirred at 37 ° C. for 18 minutes. After the reaction, the streptavidin-bound magnetic particle suspension (30 μL) and the alkaline phosphatase-labeled competing substance solution (30 μL) of Example 1 were added to the reaction solution of the reaction, stirred, and reacted at 37 ° C. for 8 minutes. rice field. After collecting the magnetic particles by magnetic force and removing the reaction solution other than the magnetic particles, a cleaning solution [50 mmol / L MOPS buffer (pH 7.35) containing 0.1% tween 20] is added to the reaction vessel, and the magnetic particles are added. Was washed. The magnetic particles were collected by a magnetic force to remove a solution other than the magnetic particles, and then a cleaning solution was added to the reaction vessel to wash the magnetic particles. After performing a series of operations of this collection of magnetism, removal of solutions other than magnetic particles, and cleaning of magnetic particles with a cleaning solution five times, 9- (4-chlorophenylthiophosphoryloxymethylidene) -10-methylacridane / disodium A luminescent substrate solution (70 μL) containing a salt as a main component was added and stirred, and the generated luminescence amount (RLU) was measured to prepare a calibration curve 1 showing the relationship between the aldosterone concentration and the luminescence amount (RLU).

Instead of each aldosterone standard solution, biotin-binding anti-aldosterone antibody solution, streptavidin-binding magnetic particle suspension, and alkaline phosphatase-labeled competitor solution of the kit of Example 1, each aldosterone standard solution of Comparative Example 1, biotin binding A calibration line 2 showing the relationship between the aldosterone concentration and the luminescence amount (RLU) was obtained by the same method as described above except that the anti-aldosterone antibody solution, the streptavidin-bound magnetic particle suspension, and the alkaline phosphatase-labeled competing substance solution were used. Created.

(2) Determination of aldosterone concentration in sample-correlation test Instead of each aldosterone standard solution of Example 1 and Comparative Example 1, 13 serum or plasma samples collected from patients with primary aldosteronism and healthy subjects. The amount of luminescence (RLU) for each of the 13 samples was measured by the same method as in (1) except that. The aldosterone concentration in each sample was determined from the luminescence amount (RLU) obtained for each sample and the calibration curve prepared in (1). When determining the concentration of aldoteron using the kit of Example 1, a calibration curve 1 is used as a calibration curve, and when determining the concentration of aldoteron using the kit of Comparative Example 1, a calibration curve is used as a calibration curve. 2 was used.

In addition, using a commercially available aldosterone measurement kit "Spack-S aldosterone kit" (manufactured by Fujirebio), measurement was performed according to the measurement procedure described in the package insert of the kit, and the aldosterone concentration in each of the 13 samples was measured. It was determined.
Correlation test is one of the methods for determining whether or not accurate measurement is possible. In the test, a plurality of identical samples are measured using an existing in-vitro diagnostic drug and an evaluation reagent to be compared with the in-vitro diagnostic drug, and the measured values obtained by the measurement using each reagent are used. , Statistical calculation of correlation equation (Y = aX + b) and correlation coefficient (r), and evaluation of the results. As the slope "a" in the correlation equation is closer to 1.0 and the correlation coefficient (r) is closer to 1.0, the measured value in the existing in vitro diagnostic drug and the measured value in the evaluation reagent are the same and more accurate. It shows that the measurement is possible.

The slope of the correlation equation between the measurement of aldosterone using the kit of Example 1 or the kit of Comparative Example 1 and the measurement of aldosterone using the "Spack-S aldosterone kit" (manufactured by Fujirebio), and the correlation coefficient. Is shown in Table 1.

As is clear from Table 1, the measurement of aldosterone using each of the kits a to c, that is, the measurement of aldosterone using only the nonionic surfactant and not the buffer (I) is described in "Spack-". The slopes of the correlation equation with the measurement of aldosterone using "S aldosterone kit" (manufactured by Fujirebio) are 0.80, 0.81 and 1.37, and accurate measurement is not possible. The measurement of aldosterone used, that is, the measurement of aldosterone of the present invention using the buffer (I) and the nonionic surfactant, is between the measurement of aldosterone using "Spack-S aldosterone kit" (manufactured by Fujirebio). The slope of the correlation equation was 0.96 to 1.05, which was close to 1.0, and the correlation coefficient was 0.97 to 0.99, which was close to 1.0. Therefore, it was found that the method for measuring aldosterone of the present invention using the buffer (I) and the nonionic surfactant is a method capable of accurately measuring aldosterone.

The cross-reactivity of corticosterone, cortisol and cortisone to each cross-reactive substance in the measurement of aldosterone using the kits A to E of Example 1 and the kits a to c of Comparative Example 1 was evaluated. Corticosterone, cortisol and cortisone are all steroid hormones similar in structure to aldosterone.
Each cross-reactive substances, degreasing treated plasma was prepared by diluting with [Human Plasma, Defibrinated, Delipidized double charcoal stripped ( manufactured by Golden West Biologicals, Inc.)], 10~250μg / mL (= 1 × 10 7 ~2.5 Using each cross-reactive substance solution (× 10 ⁸ pg / mL) as a sample, measurement was carried out according to the method (2) of Example 2, and the cross-reactivity rate for each cross-reactive substance was determined by the following formula. .. The results are shown in Table 2.

Since the cross-reactivity is an index showing the rate at which cross-reactive substances that should not be measured are measured, the lower the cross-reactivity, the more specificly aldosterone can be detected, and the more accurate the measurement can be. It shows that it is.

As is clear from Table 2, the kit is compared to the measurement of aldosterone using each of the kits b to c, that is, the measurement of aldosterone using only the nonionic surfactant and not the buffer (I). In the measurement of aldosterone using each of the kits A to E, that is, in the measurement of aldosterone using the buffer (I) and the nonionic surfactant, the cross-reactivity to each cross-reactive substance was suppressed. Therefore, it was found that the method for measuring aldosterone of the present invention using the buffer (I) and the nonionic surfactant is a method capable of accurately measuring aldosterone.

Kits F to Q for measuring aldosterone consisting of the following streptavidin-bound magnetic particle suspension, biotin-bound anti-aldosterone antibody solution, alkaline phosphatase-labeled competitor solution, and aldosterone standard solution were prepared.

<Streptavidin-bonded magnetic particle suspension>
As the magnetic particles, commercially available magnetic particles (Dynabeads M-280 Streptavidin: manufactured by Thermo Fisher Scientific Co., Ltd.) to which streptavidin is bound are used, and a streptavidin-bound magnetic particle suspension (pH 6.5) having the following composition is used. Was prepared.

BES 125 mmol / L
Streptavidin-bonded magnetic particles 0.7 mg / mL
BSA 0.1%
Surfactants (see Table 3)

<Biotin-bound anti-aldosterone antibody solution>
Using the anti-aldosterone polyclonal antibody obtained in the reference example, and using the biotin-binding anti-aldosterone antibody obtained in the same manner as in Example 1, a biotin-binding anti-aldosterone antibody solution (pH 6.5) having the following composition. Was prepared.

BES 125 mmol / L
Biotin-binding anti-aldosterone antibody 22.5 ng / mL
BSA 0.1%

<Alkaline phosphatase-labeled competing substance solution>
An alkaline phosphatase-labeled competing substance solution (pH 7.5) having the following composition was prepared using the alkaline phosphatase-labeled competing substance prepared in the same manner as in Example 1.

TES 20 mmol / L
Alkaline phosphatase-labeled competitor 1.5 μg / mL
BSA 0.1%
Surfactants (see Table 3)
Magnesium chloride hexahydrate 30 mmol / L

<Aldosterone standard solution>
An aldosterone solution having an aldosterone concentration of 0, 16, 40, 80, 160, 400, 1600 pg / mL was prepared in the same manner as in Example 1, and the solution was used as an aldosterone standard solution for preparing a calibration curve. bottom.

As the surfactant in the streptavidin-bonded magnetic particle suspension and the surfactant in the alkaline phosphatase-labeled competing substance solution, the same type and concentration were used.

[Comparative Example 2]
Instead of the streptavidin-bound magnetic particle suspension and the alkali phosphatase-labeled competing substance solution of the kit of Example 4, the streptavidin-binding magnetic particle suspension containing no surfactant and the alkali phosphatase-labeled competing substance A kit consisting of a surfactant-free streptavidin-bound magnetic particle suspension, a biotin-bound anti-aldosterone antibody solution, a surfactant-free alkaline phosphatase-labeled competing substance solution, and the aldosterone standard solution of Example 4 prepared as a solution. d was prepared.

(1) Preparation of calibration line To the reaction vessel, each aldosterone standard solution (10 μL) of Example 4 and the biotin-binding anti-aldosterone antibody solution (50 μL) of Example 4 were added and stirred, and the mixture was stirred at 37 ° C. for 18 minutes. After the reaction, the streptavidin-bound magnetic particle suspension (30 μL) and the alkaline phosphatase-labeled competing substance solution (30 μL) of Example 4 were added to the reaction solution of the reaction, stirred, and reacted at 37 ° C. for 8 minutes. rice field. After collecting the magnetic particles by magnetic force and removing the reaction solution other than the magnetic particles, a cleaning solution [50 mmol / L MOPS buffer (pH 7.35) containing 0.1% tween 20] is added to the reaction vessel, and the magnetic particles are added. Was washed. The magnetic particles were collected by a magnetic force to remove a solution other than the magnetic particles, and then a cleaning solution was added to the reaction vessel to wash the magnetic particles. After performing a series of operations of this collection of magnetism, removal of solutions other than magnetic particles, and cleaning of magnetic particles with a cleaning solution five times, 9- (4-chlorophenylthiophosphoryloxymethylidene) -10-methylacridane / disodium A luminescent substrate solution (70 μL) containing a salt as a main component was added and stirred, and the generated luminescence amount (RLU) was measured to prepare a calibration curve 3 showing the relationship between the aldosterone concentration and the luminescence amount (RLU).

Instead of each solution or suspension of the aldosterone standard solution, the biotin-conjugated anti-aldosterone antibody solution, the streptavidin-conjugated magnetic particle suspension, and the alkaline phosphatase-labeled competitor solution of the kit of Example 4, the aldosterone of Comparative Example 2 Aldosterone concentration and luminescence amount by the same method as described above except that each solution or suspension of a standard solution, a biotin-bonded anti-aldosterone antibody solution, a streptavidin-bound magnetic particle suspension, and an alkaline phosphatase-labeled competing substance solution is used. A calibration line 4 showing the relationship with (RLU) was created.

(2) Determination of aldosterone concentration in sample-correlation test Instead of each aldosterone standard solution of Example 4 and Comparative Example 2, 13 serum or plasma samples collected from patients with primary aldosteronism and healthy subjects. The amount of luminescence (RLU) for each of the 13 samples was measured by the same method as in (1) except that. The aldosterone concentration in each sample was determined from the luminescence amount (RLU) obtained for each sample and the calibration curve prepared in (1). When determining the concentration of aldoteron using the kit of Example 4, a calibration curve 3 is used as a calibration curve, and when determining the concentration of aldoteron using the kit of Comparative Example 2, a calibration curve is used as a calibration curve. 4 was used.

In addition, using a commercially available aldosterone measurement kit "Spack-S aldosterone kit" (manufactured by Fujirebio), measurement was performed according to the measurement procedure described in the package insert of the kit, and the aldosterone concentration in each of the 13 samples was measured. It was determined.
Measurement of aldosterone using the streptavidin-bound magnetic particle suspension, biotin-bound anti-aldosterone antibody solution, and alkaline phosphatase-labeled competitor solution prepared in Example 4 and Comparative Example 2, and "Spack-S aldosterone kit" (Fuji) Table 3 shows the slope of the correlation equation with the measurement of aldosterone using (manufactured by Lebio) and the correlation coefficient.

As is clear from Table 3, the measurement of aldosterone using the kit of kit d, that is, the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant using only the buffer (I). In the measurement of aldosterone without using any of the surfactants, the slope of the correlation equation between the measurement of aldosterone using the "Spack-S aldosterone kit" (manufactured by Fujirebio) is 1.30, and accurate measurement cannot be performed. On the other hand, the measurement of aldosterone using each of the kits F to Q, that is, the buffer (I), and the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant. In the measurement of aldosterone of the present invention using at least one selected from the group, the slope of the correlation equation with the measurement of aldosterone using the "Spack-S aldosterone kit" (manufactured by Fujirebio) is 0.86 to 1.21. It was close to 1.0, and the correlation coefficient was 0.97 to 1.00, which was close to 1.0. Therefore, the method for measuring aldosterone of the present invention using the buffer (I) and at least one selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant is used. It turned out to be a method that can accurately measure aldosterone.

The cross-reactivity of progesterone, corticosterone, cortisol and cortisone to the cross-reactive substances in the measurement of aldosterone using the kits F to Q of Example 4 and the kit d of Comparative Example 2 was evaluated. Progesterone, corticosterone, cortisol and cortisone are all steroid hormones similar in structure to aldosterone.

Each cross-reactive substances, degreasing treated plasma was prepared by diluting with [Human Plasma, Defibrinated, Delipidized double charcoal stripped ( manufactured by Golden West Biologicals, Inc.)], 10~250μg / mL (= 1 × 10 7 ~2.5 Using each cross-reactive substance solution (× 10 ⁸ pg / mL) as a sample, measurement was performed according to the method (2) of Example 5, and the cross-reactivity rate for each cross-reactive substance was measured by the same formula as in Example 3. It was determined. The results are shown in Table 4.

As is clear from Table 4, the measurement of aldosterone using kit d, that is, any of nonionic surfactants, anionic surfactants, and amphoteric surfactants using only buffer (I). Compared to the measurement of aldosterone without using a surfactant, the measurement of aldosterone using each of the kits F to Q, that is, the buffer (I), the nonionic surfactant, and the anionic surfactant In the measurement of aldosterone using at least one surfactant selected from the group consisting of amphoteric surfactants, cross-reactivity to each cross-reactive substance was suppressed. Therefore, the aldosterone of the present invention using the buffer (I) and at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. The measuring method was found to be a method capable of accurately measuring aldosterone.

INDUSTRIAL APPLICABILITY The present invention provides a method for measuring a steroid hormone in a sample, a reagent for measuring, and a kit for measuring, which are useful for clinical diagnosis.

Claims (14)

Specimens and anti-aldosterone antibodies and labeling competitors
3-Morphorinopropanesulfonic acid (MOPS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And a buffer selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), and
Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
Aldosterone in a sample is characterized by reacting in an aqueous medium containing aldosterone to generate an immune complex of an anti-aldosterone antibody and a labeled competing substance, and measuring the label in the generated immune complex. Measurement method. The measuring method according to claim 1, wherein a labeled competing substance is added to the reaction solution of the reaction after reacting the sample with the anti-aldosterone antibody. The measuring method according to claim 1 or 2, wherein the anti-aldosterone antibody is immobilized on an insoluble carrier. Specimens and labeled anti-aldosterone antibodies and competing substances
3-Morphorinopropanesulfonic acid (MOPS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And a buffer selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), and
Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
Aldosterone in a sample, which comprises reacting in an aqueous medium containing the aldosterone to generate an immune complex of a labeled anti-aldosterone antibody and a competitor, and measuring the label in the generated immune complex. Measurement method. The measuring method according to claim 4, wherein after reacting the sample with the labeled anti-aldosterone antibody, a competing substance is added to the reaction solution of the reaction. The measuring method according to claim 4 or 5, wherein the competing substance is immobilized on an insoluble carrier. Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
3-Morphorinopropanesulfonic acid (MOPS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And a buffer selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris),
Labeling competitors, as well as
Contains anti-aldosterone antibody,
A reagent for measuring aldosterone in a sample. The reagent according to claim 7, wherein the anti-aldosterone antibody is immobilized on an insoluble carrier. Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
3-Morphorinopropanesulfonic acid (MOPS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And a buffer selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris),
Labeled anti-aldosterone antibody, as well as
Including competing substances,
A reagent for measuring aldosterone in a sample. The reagent according to claim 9, wherein the competing substance is immobilized on an insoluble carrier. It contains a first reagent containing an anti-aldosterone antibody and a second reagent containing a labeling competitor.
Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
3-Morphorinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And each of the buffers selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) is contained in either or both of the first reagent and the second reagent. Kit for measuring aldosterone inside. The kit according to claim 11, wherein the anti-aldosterone antibody is immobilized on an insoluble carrier. It contains a first reagent containing a labeled anti-aldosterone antibody and a second reagent containing a competing substance.
Nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polycyclic phenyl ethers, polyoxyethylene polyoxypropylene condensates and polyoxyethylene alkyl amines,
Anionic surfactants selected from cole acids, and
At least one surfactant selected from the group consisting of amphoteric surfactants,
3-Morphorinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) ), And each of the buffers selected from the group consisting of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) is contained in either or both of the first reagent and the second reagent. Kit for measuring aldosterone inside. 13. The kit of claim 13, wherein the competitor is immobilized on an insoluble carrier.