JP2017142241A - Method of measuring steroid hormones in sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of accurately measuring steroid hormones in a sample without being affected by cross-reactive substances.SOLUTION: A method of measuring steroid hormones in a sample involves; having a sample, anti-steroid hormone antibody, and labeling competitive substance react with each other in an aqueous medium containing a buffer agent having a structure represented by a general formula (I) (in the formula, X represents a hydroxyl group or sulfo group, and n represents an integer in a range of 2 to 5), and at least one surfactant selected from a group consisting of a nonionic surfactant, anionic surfactant, and ampholytic surfactant to produce an immune complex of the anti-steroid hormone antibody and the labeling competitive substance; and measuring the labeling substance contained in the produced immune complex.SELECTED DRAWING: None

Description

  The present invention relates to a method for measuring steroid hormones in a specimen, a measuring reagent, and a measuring kit.

  Steroid hormones are hormones having a steroid skeleton having a molecular weight of 300 to 400, 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 the body have been studied, gas chromatography-mass spectrometry (GC-MS) method, liquid chromatography-mass spectrometry (LC-MS) Methods, immunoassays, etc. have been developed. The GC-MS method and LC-MS method have high analytical performance, but the analyzers used for these analyzes are expensive and have problems such as complicated operations such as concentration and extraction operations. . On the other hand, immunoassays are often used in clinical research because they are simple and can measure steroid hormones in a short time.

  As immunological measurement methods, the sandwich method, the competitive method, etc. are known. Usually, the sandwich method is used when measuring a macromolecule such as a protein, and a low molecular weight compound such as a steroid hormone is used. Is used as a measurement object, the competition method is used. However, in the measurement of steroid hormones by the competitive method, the sensitivity and specificity are often due to the “cross-reaction” in which the antibody used reacts with steroid hormones other than the measurement target substance having a structure similar to that of the measurement target substance. 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 And a method for measuring the low molecular weight compound using a low molecular weight compound bound to a labeling substance via a linker has been reported (see Patent Document 1).

  Aldosterone is a mineral steroid hormone having a molecular weight of about 360 that is secreted from the globular layer belonging to the outer layer of the adrenal cortex, and its secretion mainly depends on the renin-angiotensin-aldosterone system. Aldosterone is a vital hormone for maintaining electrolyte homeostasis, circulating blood volume, and blood pressure, acting on the mineralocorticoid receptor in the distal renal tubule to promote water reabsorption along with sodium reabsorption It has a function to increase blood pressure.

  Aldosterone is used for differential diagnosis of primary aldosteronism, Barter syndrome, Riddle syndrome, hydroxylase deficiency such as 17α hydroxylase deficiency in adrenal gland, 11β hydroxylase deficiency, and selective hypoaldosteronism Has been. In addition, in the Japan Endocrine Society Guidelines and the Japan Hypertension Society Guidelines, as a screening method for primary aldosteronism, renin activity (Plasma Renin Activity: PRA) or renin concentration (Active Renin Concentration: PAC) : Measurement of Aldosterone to Renin Ratio (ARR) which is a ratio to ARC is recommended.

  Known methods for measuring aldosterone include the LC-MS method and immunoassay, and examples of the immunoassay include radioimmunoassay (RIA) and enzyme immunoassay (EIA). (See Patent Document 2). However, when aldosterone in plasma of patients with chronic renal failure is measured by the RIA method, the steroid hormone reacts with the anti-aldosterone antibody because there is a large amount of steroid hormone similar in structure to aldosterone in the sample. It has been reported that an accurate measurement value cannot be obtained due to the influence of cross reaction (see Non-Patent Document 1).

JP 2013-083448 A JP-A-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. There is to do.

  The present inventors have intensively studied to solve this problem, and in the measurement by a competitive method for steroid hormones in a specimen, the antigen-antibody reaction includes a buffer having a specific structure and a specific surfactant. As a result, the inventors have found that cross-reactions are suppressed and accurate steroid hormone measurement is possible by carrying out in an aqueous medium, and the present invention has been completed. That is, the present invention relates to the following [1] to [23].

[1] A specimen, an antisteroid hormone antibody, and a labeled competitor are represented by the general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), 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 the antisteroid hormone antibody and the labeled competitor, A method for measuring a steroid hormone in a specimen, which comprises measuring a label in an immune complex.
[2] The measurement method according to [1], wherein the sample is reacted with the antisteroid hormone antibody, and then a labeled competitor is added to the reaction solution.
[3] The measurement method according to [1] or [2], wherein the antisteroid hormone antibody is immobilized on an insoluble carrier.
[4] A specimen, a labeled antisteroid hormone antibody and a competitor are represented by the general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a nonionic surfactant, an anionic surfactant, and Reacting in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to generate an immune complex between the labeled antisteroid hormone antibody and the competitor, A method for measuring a steroid hormone in a specimen, which comprises measuring a label in an immune complex.
[5] The measurement method according to [4], wherein the sample is reacted with the labeled antisteroid hormone antibody, and then a competitive substance is added to the reaction solution of the reaction.
[6] The measurement method according to [4] or [5], wherein the competitive substance is immobilized on an insoluble carrier.
[7] The buffer is 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES), and bis (2-hydroxyethyl) iminotris The measurement 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 measurement method according to any one of [1] to [7], wherein the steroid hormone is a mineral corticoid.
[9] The measurement method according to [8], wherein the mineral corticoid is aldosterone.

[10] At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5) in a sample containing a buffer having a structure represented by: Reagent for measuring steroid hormones.
[11] The reagent according to [10], wherein the antisteroid 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)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a buffering agent having a structure represented by: a labeled antisteroid hormone antibody, and a competitor, Reagent for measuring steroid hormones.
[13] The reagent according to [12], wherein the competitive substance is immobilized on an insoluble carrier.
[14] The buffer is 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (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 mineral corticoid.
[16] The reagent according to [15], wherein the mineral corticoid is aldosterone.

[17] Contains a first reagent containing an antisteroid hormone antibody and a second reagent containing a labeled competitor, and consists of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant At least one surfactant selected from the group, and the general formula (I)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), each of the buffering agents is included in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in a specimen.
[18] The kit according to [17], wherein the antisteroid hormone antibody is immobilized on an insoluble carrier.
[19] Contains a first reagent containing a labeled antisteroid hormone antibody and a second reagent containing a competitor, and consists of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant At least one surfactant selected from the group, and the general formula (I)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), each of the buffering agents is included in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in a specimen.
[20] The kit according to [19], wherein the competitive substance is immobilized on an insoluble carrier.
[21] The buffer is 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N , N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (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 mineral corticoid.
[23] The kit according to [22], wherein the mineral corticoid is aldosterone.

  According to the present invention, there are provided a method, a reagent, and a kit that enable accurate measurement of a steroid hormone in a specimen without being affected by a cross-reactive substance.

(Measuring method)
The method for measuring steroid hormone in a sample of the present invention comprises a sample, an antisteroid hormone antibody and a labeled competitor substance, which are represented by the general formula (I):

(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5) and a nonionic surfactant having a structure represented by the following formula (hereinafter referred to as buffer (I)) Immunization between an antisteroid hormone antibody and a labeled competitor by reacting in an aqueous medium containing at least one surfactant selected from the group consisting of an anionic surfactant and an amphoteric surfactant It is a method characterized by producing a complex and measuring the label in the produced immune complex.

As one embodiment (embodiment 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 specimen, the antisteroid hormone antibody and the labeled competitor are selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an 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 The step of causing;
[2] A step of measuring the labeling substance in the immune complex of the antisteroid hormone antibody and the labeled competitor substance produced in the step [1];
[3] A step of performing a step [1] and a step [2] using a steroid hormone having a known concentration instead of a specimen to create a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[4] A step of determining a steroid hormone concentration in the sample from the calibration curve created in the step [3] and the measured value of the labeling substance measured in the step [2].

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

As another aspect (aspect 2) of the measuring method of the steroid hormone of this invention, the method including the following processes is mentioned, for example.
[1] At least one surfactant selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. Reacting in an aqueous medium containing an agent;
[2] A step of adding a labeled competitor to the reaction solution of step [1];
[3] A step of measuring the labeling substance in the immune complex of the antisteroid hormone antibody and the labeled competitor substance produced in the step [2];
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

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

As another embodiment (embodiment 3) of the method for measuring steroid hormones of the present invention, for example, a method including the following steps can be mentioned.
[1] A step of reacting a specimen and an antisteroid hormone antibody in an aqueous medium containing the buffer (I);
[2] At least one surfactant selected from the group consisting of a labeled competitor, a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant is added to the reaction solution of step [1]. Adding the agent;
[3] A step of measuring the labeling substance in the immune complex of the antisteroid hormone antibody and the labeled competitor substance produced in the step [2];
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

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

Another embodiment (embodiment 4) of the method for measuring steroid hormones of the present invention includes, for example, a method comprising the following steps.
[1] An aqueous medium containing a specimen and an antisteroid hormone antibody containing at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant Reacting in;
[2] A step of adding a labeled competitor and a buffering agent (I) to the reaction solution of step [1];
[3] A step of measuring the labeling substance in the immune complex of the antisteroid hormone antibody and the labeled competitor substance produced in the step [2];
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

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

Furthermore, as another embodiment (embodiment 5) of the method for measuring steroid hormones of the present invention, for example, a method including the following steps may be mentioned.
[1] A step of reacting a specimen and an antisteroid hormone antibody in an aqueous medium;
[2] The reaction solution of step [1] is selected from the group consisting of a labeled competitor, buffer (I), and a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. Adding at least one surfactant as described above;
[3] A step of measuring the labeling substance in the immune complex of the antisteroid hormone antibody and the labeled competitor substance produced in the step [2];
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

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

When the antisteroid hormone antibody is immobilized on an insoluble carrier, the insoluble carrier on which the antisteroid hormone antibody is immobilized may be produced in a reaction solution of an antigen-antibody reaction. In this case, a set of affinity substances By reacting an anti-steroid hormone antibody to which one of (A) is bound and an insoluble carrier to which the other (a) of a pair of affinity substances is bound in a reaction solution for antigen-antibody reaction, anti-steroid hormone antibody Can be produced in the reaction solution for the antigen-antibody reaction. Examples of the combination of A-a include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-Combinations of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
A combination of an Fc region of an antisteroid hormone antibody and an antibody that binds to the Fc region.

  Reaction of specimen and antisteroid hormone antibody in step [1] in aspect 1 and steps [1] and step [2] in aspects 2 to 5, and reaction of reaction between labeled competitor and antisteroid 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, particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, and preferably 2 minutes to 1 hour.

  In Step [1] in Embodiments 1 to 5, the specimen can be subjected to a reaction after pretreatment with an aqueous medium described later in advance. Examples of the pretreatment include dilution, solubilization, and denaturation. The aqueous medium may contain metal ions, saccharides, preservatives, proteins and the like described later. The temperature at which the specimen is pretreated with an aqueous medium in advance 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., preferably 20 to 40 ° C. is particularly preferred. The time for pretreating the specimen in advance with an aqueous medium is not particularly limited as long as it allows 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 labeled substance in step [2] in aspect 1 and step [3] in aspects 2 to 5 is not particularly limited as long as it is a method enabling measurement of the steroid hormone of the present invention. The method etc. are mentioned.

  The method for measuring steroid hormone in a sample of the present invention also comprises a sample, a labeled antisteroid hormone antibody and a competitor, a buffer (I), a nonionic surfactant, and an anionic surfactant. And reacting in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to form an immune complex of a labeled antisteroid hormone antibody and a competitor And measuring the labeling substance in the immune complex.

As one embodiment (embodiment 6) of the method for measuring steroid hormones of the present invention, for example, a method comprising the following steps can be mentioned.
[1] The specimen, the labeled antisteroid hormone antibody and the competitor are selected from the group consisting of a buffer (I), a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. And an immunocomplex of the labeled antisteroid hormone antibody and the steroid hormone, and an immunocomplex of the labeled antisteroid hormone antibody and the competitor by reacting in an aqueous medium containing at least one surfactant. Generating a body;
[2] A step of measuring the labeling substance in the immune complex of the labeled antisteroid hormone antibody and the competitor substance produced in step [1];
[3] A step of performing a step [1] and a step [2] using a steroid hormone having a known concentration instead of a specimen to create a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance;
[4] A step of determining a steroid hormone concentration in the sample from the calibration curve created in the step [3] and the measured value of the labeling substance measured in the step [2].

  The competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

Another embodiment (embodiment 7) of the method for measuring steroid hormones of the present invention includes, for example, a method comprising the following steps.
[1] Sample and labeled antisteroid hormone antibody, at least one selected from the group consisting of buffer (I), nonionic surfactant, anionic surfactant, and amphoteric surfactant Reacting in an aqueous medium containing a surfactant of:
[2] A step of adding a competitive substance to the reaction solution of step [1];
[3] A step of measuring the labeling substance in the immune complex of the labeled antisteroid hormone antibody produced in step [2] and the competitor;
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

  The competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

Another embodiment (embodiment 8) of the method for measuring steroid hormones of the present invention includes, for example, a method comprising the following steps.
[1] A step of reacting a specimen and a labeled antisteroid hormone antibody in an aqueous medium containing a buffer (I);
[2] The reaction liquid in step [1] contains at least one surfactant selected from the group consisting of a competing substance, a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. Adding
[3] A step of measuring the labeling substance in the immune complex of the labeled antisteroid hormone antibody produced in step [2] and the competitor;
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

  The competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

Another embodiment (embodiment 9) of the method for measuring steroid hormones of the present invention includes, for example, a method comprising the following steps.
[1] The specimen 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. Reacting in an aqueous medium;
[2] A step of adding a competitive substance and a buffer (I) to the reaction solution of step [1];
[3] A step of measuring the labeling substance in the immune complex of the labeled antisteroid hormone antibody produced in step [2] and the competitor;
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

  The competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

Furthermore, another embodiment (embodiment 10) of the method for measuring steroid hormones of the present invention includes, for example, a method comprising the following steps.
[1] A step of reacting a specimen with a labeled antisteroid hormone antibody in an aqueous medium;
[2] At least selected from the group consisting of competitor, buffer (I), and nonionic surfactant, anionic surfactant, and amphoteric surfactant in the reaction solution of step [1] Adding one surfactant;
[3] A step of measuring the labeling substance in the immune complex of the labeled antisteroid hormone antibody produced in step [2] and the competitor;
[4] A step of creating a calibration curve representing the relationship between the steroid hormone concentration and the measured value of the labeling substance by performing the steps [1] to [3] using a known concentration of steroid hormone instead of the specimen;
[5] A step of determining the steroid hormone concentration in the specimen from the calibration curve created in the step [4] and the measured value of the labeled substance measured in the step [3].

  The competitor may or may not be immobilized on an insoluble carrier, but is preferably immobilized.

When the competitive substance is immobilized on the insoluble carrier, the insoluble carrier on which the competitive substance is immobilized may be generated in the reaction solution of the antigen-antibody reaction. In this case, one of the pair of affinity substances (B ) And the insoluble carrier to which the other (b) of the pair of affinity substances are bound in the reaction solution of the antigen-antibody reaction, the insoluble carrier having the competitor immobilized thereon is reacted. It can be produced in a reaction solution for antigen-antibody reaction. Examples of the combination of B-b include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
A combination of avidin (such as avidin, neutravidin, streptavidin, etc.) and biotin.

  Reaction of specimen and labeled antisteroid hormone antibody in step [1] in aspect 6 and steps [1] and [2] in aspects 7 to 10, and reaction between competitor and labeled antisteroid hormone antibody The reaction 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, particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables measurement of the steroid hormone of the present invention, and is usually 1 minute to 2 hours, and preferably 2 minutes to 1 hour.

  In Step [1] in Embodiments 6 to 10, the specimen can be subjected to a reaction after pretreatment with an aqueous medium described later in advance. Examples of the pretreatment include dilution, solubilization, and denaturation. The aqueous medium may contain metal ions, saccharides, preservatives, proteins and the like described later. The temperature at which the specimen is pretreated with an aqueous medium in advance 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., preferably 20 to 40 ° C. is particularly preferred. The time for pretreating the specimen in advance with an aqueous medium is not particularly limited as long as it allows 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 labeled substance in step [2] in aspect 6 and step [3] in aspects 7 to 10 is not particularly limited as long as it is a method enabling measurement of the steroid hormone of the present invention. The method etc. are mentioned.

  The steroid hormone in the present invention is not particularly limited as long as it is a steroid hormone that enables the measurement of the present invention. Examples thereof include mineralocorticoids, glucocorticoids, sex hormones, and the like, and mineralocorticoids are preferable. Examples of mineral corticoids include aldosterone and fludrocortisone acetate. Examples of glucocorticoids include cortisol, corticosterone, cortisone and the like. Examples of sex hormones include male hormones (androgens) and female hormones. Examples of male hormones include testosterone and dehydroepiandrosterone. 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, amniotic fluid, sweat, pancreatic juice and the like. 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), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES). 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (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 (EPP ), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and the like. 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N-bis (2-Hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) is preferred.

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

  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 nonionic surfactant, a nonionic surfactant, Examples include polyoxyethylene nonionic surfactants, and polyoxyethylene nonionic surfactants are preferred. Examples of the polyoxyethylene nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polyoxypropylene condensate, polyoxyethylene alkylamine, Examples include alkylene diamine-polyoxyethylene polyoxypropylene condensate, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of non-polyoxyethylene nonionic surfactants include sorbitan fatty acid esters, glycerin fatty acid esters, fatty acid alkanolamides, and sucrose fatty acid esters.

  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 dibenzyl phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene (distyrenated) phenyl ether, polyoxyethylene (tristyrenated) phenyl ether, and the like. It is 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 ethylenediaminetetrapolyoxyethylene / polyoxypropylene / block polymer.

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

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

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

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

  Examples of fatty acid alkanolamides include lauric acid diethanolamide. Examples of the sucrose fatty acid ester include sucrose palmitate ester and sucrose stearate ester.

  Examples of the anionic surfactant include carboxylate, sulfonate, sulfate ester salt, phosphate ester salt, and cholic acid.

  Examples of the carboxylate include salts with 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 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 acid such as dodecylbenzene sulfonic acid, naphthalene sulfonic acid such as dipropylnaphthalene sulfonic acid and dibutyl naphthalene sulfonic acid, sulfosuccinic acid such as dioctyl sulfosuccinic acid, and a salt with sodium. Can be mentioned. Specific examples include sodium dodecylbenzenesulfonate, sodium dipropylnaphthalenesulfonate, sodium dibutylnaphthalenesulfonate, sodium dioctylsulfosuccinate and the like.

  Examples of sulfate salts include higher alcohol sulfate salts, polyoxyethylene alkyl ether sulfate salts, polyoxyethylene polycyclic phenyl ether sulfate salts, and the like. Examples of the salt include sodium salt and ammonium salt. 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 and the like. Specific examples of the cyclic phenyl ether sulfate ester salt include sodium polyoxyethylene polycyclic phenyl ether sulfate.

  Examples of phosphoric acid ester salts include monoalkyl phosphoric acid ester salts and polyoxyethylene alkyl ether phosphoric acid ester salts. Examples of the salt include sodium salt and potassium salt. Specific examples of monoalkyl phosphate salts include, for example, sodium monostearyl phosphate, sodium monododecyl phosphate, and the like. Specific examples of polyoxyethylene alkyl ether phosphate salts include, for example, polyoxyethylene dodecyl ether phosphate. Potassium etc. are mentioned.

  Examples of cholic acids include cholic acid, taurocholic acid, glycocholic acid, lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, 7-oxolithocholic acid, 12-oxolithocholic acid, 12-oxochenodeoxycholic acid, 7 -Oxodeoxycholic acid, hyocholic acid, hyodeoxycholic acid, dehydrocholic acid and the like. These cholic acids may form a salt. 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, bile acids and the like.

  Examples of betaines include alkylbetaines, carboxybetaines, and sulfobetaines. Examples of the alkylbetaines include N-dodecylbetaine. Examples of carboxybetaines include amidopropyl betaine laurate, betaine dodecyldimethylaminoacetate, sodium N-lauroyl-N′-carboxymethyl-N′-hydroxyethylethylenediamine, and the like. Examples of the sulfobetaines include lauric acid amidopropyl hydroxysulfobetaine.

  Examples of glycines include sodium dodecyldiaminoethylglycine.

Examples of alanines include sodium N-octodecyl-β-alanine, sodium N-mystyryl-β-alanine, sodium laurylaminopropionate, and the like.
Examples of the 2-alkylimidazoline derivative include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine such as 2-lauroyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine.

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

  Examples of the alkylamino salts include sodium dodecylaminodiacetate.

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

  The concentration of the nonionic surfactant, the anionic surfactant or the amphoteric surfactant in the reaction solution of the reaction between the analyte or the labeled competitor and the antisteroid hormone antibody is the steroid hormone of the present invention. The concentration is not particularly limited as long as it can be measured, and is usually 0.005 to 3%, preferably 0.01 to 2%. In addition, the concentration of the nonionic surfactant, the anionic surfactant, or the amphoteric surfactant in the reaction solution of the reaction between the specimen or the competitor and the labeled antisteroid hormone antibody may be 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 measurement 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 competitor and enables measurement of the steroid hormone of the present invention. For example, a polystyrene plate such as a microtiter plate, glass, etc. Examples thereof include granular products (beads) made of synthetic resin or synthetic resin, spherical materials (ball) made of glass or synthetic resin, latex, magnetic particles, various membranes such as nitrocellulose membrane, synthetic resin test tubes, and the like.

  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 that enables measurement of the steroid hormone of the present invention, and examples thereof include physical adsorption and chemical bond. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonds include covalent bonds and coordinate bonds.

  The antisteroid hormone antibody may be directly immobilized on an insoluble carrier or indirectly immobilized on an insoluble carrier using the physical adsorption and / or chemical bond described above. Examples of the indirect immobilization method include a method of immobilizing an antisteroid hormone antibody on an insoluble carrier through specific binding between biotin and avidin (eg, avidin, streptavidin, neutravidin, etc.). Further, the antisteroid hormone antibody may be immobilized on an insoluble carrier by a covalent bond via a linker.

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

  In the present invention, the bond between the competitor and the insoluble carrier is not particularly limited as long as it is a bond that enables measurement of the steroid hormone of the present invention, and examples thereof include physical adsorption and chemical bond. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonds include covalent bonds and coordinate bonds.

  The competitive substance may be directly immobilized on the insoluble carrier or indirectly immobilized on the insoluble carrier by using the above-described 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 through specific binding between biotin and avidin (eg, avidin, streptavidin, neutravidin, etc.). In addition, the competitor may be immobilized on an insoluble carrier by a covalent bond via a linker.

  Examples of the linker include a molecule that can covalently bond both the functional group on the surface of the insoluble carrier and the functional group of the competitor, and the first reactive active group that can react with the functional group of the competitor, and insoluble A molecule having a second reactive group capable of reacting with a functional group on the surface of the carrier in the same molecule, wherein the first reactive group and the second reactive group are different groups is preferred. Examples of the functional group of the competitive substance and the functional group held on the surface of the insoluble carrier include the functional groups described above. Examples of the reactive group in the linker include the aforementioned reactive group.

The antisteroid hormone antibody in the present invention is not particularly limited as long as it binds to the steroid hormone and enables measurement of the steroid hormone of the present invention, and either a polyclonal antibody or a monoclonal antibody can be used. In the present invention, not only full-length antibodies but also antibody fragments can be used. Examples of antibody fragments include Fabs obtained by papain treatment of antibodies, F (ab ′) ₂ obtained by pepsin treatment, antibody fragments from which Fc portions such as Fab ′ obtained by pepsin treatment-reduction treatment have been removed, etc. Can be mentioned. When using an insoluble carrier on which avidins (avidin, neutravidin, streptavidin, etc.) are immobilized, use a biotin-conjugated antisteroid hormone antibody in which biotin is bound to an antisteroid hormone antibody. Can do. In addition, when an insoluble carrier on which biotin is immobilized is used as an insoluble carrier, an avidin-binding antisteroid hormone antibody in which an avidin (eg, avidin, neutravidin, streptavidin, etc.) is bound to an antisteroid hormone antibody Can be used.

  The antisteroid hormone antibody in the present invention can be produced by a conventional antibody production method using a steroid hormone itself or a compound having a partial structure corresponding to an epitope in the steroid hormone as an antigen, It is available. Examples of commercially available antisteroid hormone antibodies include antisteroid hormone polyclonal antibodies (manufactured by Abcam).

  The labeled antisteroid hormone antibody in the present invention is prepared by the method described later using the above-mentioned antisteroid hormone antibody and the labeling material described later.

  The competing substance in the present invention means a substance that binds to an antisteroid hormone antibody and the binding is competitive with the steroid hormone that is the measurement target substance, and 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 recognized by the antisteroid hormone antibody is preferable, and the binding strength to the antisteroid hormone antibody is such that the steroid hormone binds to the antibody. More preferably, the same strength as that of the steroid hormone itself is mentioned.

  When an insoluble carrier having avidins (such as avidin, neutravidin, streptavidin, etc.) immobilized thereon is used as the insoluble carrier, a biotin-binding competitor having biotin bound to the competitor can be used. When an insoluble carrier having biotin immobilized thereon is used as the insoluble carrier, an avidin-binding competitor having an avidin (eg, avidin, neutravidin, streptavidin, etc.) bound to the competitor may be used. it can.

  The competing substance in the present invention can be prepared using a known method, for example, a method described in JP-A-60-260592 or JP-A-63-060996.

  The labeled competitive substance in the present invention is produced by the method described later using the aforementioned competitive substance and the labeled substance described later.

  The labeling substance used in the labeled antisteroid hormone antibody and the labeled competitor in the present invention is not particularly limited as long as it is a labeling substance that enables measurement of the steroid hormone of the present invention. For example, an enzyme, a fluorescent substance, Examples thereof include luminescent substances, radioisotopes, 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), rhodamine B-isothiocyanate (RITC), and the like. Examples of other fluorescent substances include quantum dot (Science, 281, 2016-2018, 1998), phycobiliproteins such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein) And fluorescent proteins such as BFP (Blue fluorescent Protein).

Examples of the light-emitting substance include acridinium and its derivatives, ruthenium complex compounds, and lophine. As the ruthenium complex compound, a compound shown in Clin. Chem. 37, 9, 1534-1539, 1991, which emits light electrochemically together with an electron donor is preferable.
Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, and ¹³¹ I.

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

  The labeling of the antisteroid hormone antibody with the labeling substance and the labeling of the competitor with the labeling substance are performed via a linker between the functional group of the antisteroid hormone antibody or the competitor and the functional group of the labeling substance. Or it can carry out by the reaction which produces a covalent bond without intervention. Examples of the functional group include a carboxyl group, amino group, glycidyl group, sulfhydryl group, hydroxyl group, amide group, imino group, hydroxysuccinyl ester group, maleimide group, and isothiocyanate group.

  In labeling a competitive substance with a labeling substance, the position in the competitive substance where the competitive substance binds to the labeling substance can be appropriately selected according to the structure of the competitive substance. For example, when the steroid hormone to be measured is aldosterone, and aldosterone itself is used as the competitor, the labeling substance can be bound to the 3rd, 4th, 6th or 21st position of aldosterone.

  Examples of the bonding method without using 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 other reagents and proceeds only by mixing under neutral to weakly alkaline conditions.

  The linker may be any linker as long as it can bind the functional group of the antisteroid hormone antibody or competitor and the functional group of the labeling substance. As 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. A molecule having a first functional group capable of reacting with a carboxyl group of a competing substance and a second functional group capable of reacting with a functional group of a labeling substance in the same molecule, etc. The first functional group and the second functional group are preferably different. Examples of the functional group of the linker include the aforementioned functional groups.

  Examples of the method for chemically combining radioisotopes include the method 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 phycobiliprotein, or a polypeptide containing a tag sequence, a known genetic recombination technique (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring According to Harbor Laboratory Press, 2001), an expression vector containing DNA encoding a fusion protein of a labeling substance and an antisteroid hormone antibody is prepared, the expression vector is introduced into an appropriate host, and the host is cultured. can do. 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 each DNA by ligase reaction.

  In the present invention, the measurement of the labeled substance in the immune complex of the antisteroid hormone antibody and the labeled competitor, and the measurement of the labeled substance in the immune complex of the labeled antisteroid hormone antibody and the competitor 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 multiwell plate reader, or the like.
When the labeling substance is a fluorescent substance, the fluorescence intensity can be measured using a fluorometer, a fluorescent multiwell plate reader, or the like.
When the labeling substance is a luminescent substance, the luminescence intensity can be measured using a luminescence photometer, a luminescent multiwell plate reader or the like.
When the labeling substance is a radioisotope, the radioactivity can be measured with a scintillation counter, a γ-well counter or the like.

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

  When the enzyme is peroxidase, the peroxidase activity can be measured by, for example, an absorbance method, a fluorescence method, or the like. As a method for measuring peroxidase activity by the absorbance method, for example, peroxidase is reacted with a combination of hydrogen peroxide and an oxidative coloring chromogen as a substrate, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. The method of measuring by etc. is mentioned. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

  A leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active 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] An amine (BCMA) etc. are mentioned.

  The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinder reagent), a combination of a coupler and a phenol.

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

  Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), 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-Dimethylanily N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (3- Methylphenyl) -N'-succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -4 -Fluoro-3,5-dimethoxyaniline (F-DAOS) and the like.

  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 a fluorescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as a substrate, and the intensity of fluorescence generated by a fluorometer, a fluorescent multiwell plate reader or the like is measured. The measuring method etc. are mentioned. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

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

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

Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -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] decan] -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 Salt (CSPD ^™ ), [10-methyl-9 (10H) -acridinylidene] phenoxymethyl phosphate, disodium salt (Lumigen ^™ APS-5), and the like.

  When the enzyme is β-D-galactosidase, β-D-galactosidase activity can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method. As a method for measuring β-D-galactosidase activity by an absorbance method (colorimetric method), for example, β-D-galactosidase and its substrate are reacted, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. The measuring method etc. are mentioned. Examples of the substrate for β-D-galactosidase include o-nitrofel-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by a luminescence method, for example, a method in which β-D-galactosidase and its substrate are reacted and the luminescence intensity of the reaction solution is measured with a luminescence intensity meter, a luminescence multiwell plate reader, etc. Etc. Examples of the substrate for β-D-galactosidase include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) and similar compounds. As a method of measuring β-D-galactosidase activity by a fluorescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescent multiwell plate reader, etc. Etc. 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 luciferase activity by the luminescence method include a method of reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of the luciferase substrate include luciferin and coelenterazine.

  When the labeling substance is other than a fluorescent substance, luminescent substance, radioisotope and enzyme, a labeling substance labeled with a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, etc., which is specifically bound to the labeling substance, A fluorescent substance, a luminescent substance, or a radioactive substance that binds to a labeled anti-steroid hormone antibody in the complex or the labeled substance constituting the labeled competitive substance and labels the substance that specifically binds to the labeled substance The labeling substance can be measured by measuring an 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 avidin when the labeling substance is biotin. In addition, using a substance that specifically binds to the labeling substance (such as an antibody or avidin that specifically binds to the labeling substance), it binds to the labeling substance in the immune complex and then specifically binds to the labeling substance. An antibody that specifically binds to a constant region of an antibody that specifically binds to a labeling substance, or an antibody that specifically binds to avidins, such as a fluorescent substance, a luminescent substance, or a radioisotope Alternatively, the labeled substance can be measured by binding a substance labeled with an enzyme and measuring the fluorescent substance, the luminescent substance, the radioisotope, or the enzyme labeling these antibodies by the above-described 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 and sorbitol. Examples of the preservative in the present invention include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Procrine 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 reagent for measuring steroid hormone in the sample of the present invention is a reagent used in the method for measuring steroid hormone of the present invention, and includes a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. A reagent comprising at least one surfactant selected from the group consisting of: a buffer (I), a labeled competitor, and an antisteroid hormone antibody. The antisteroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

  In addition, the reagent for measuring steroid hormone in the specimen 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 containing a buffer (I), a labeled antisteroid hormone antibody, and a competitor. The competitor may or may not be immobilized on the insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

  The measuring reagent of the present invention may be in a lyophilized state or in a liquid state. When a measurement reagent in a lyophilized state is used, it is dissolved in an aqueous medium before measurement and made into a liquid state for 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), a labeling agent The competitor, labeled antisteroid hormone antibody, competitor, and antisteroid hormone antibody are dissolved in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like. The liquid measurement reagent includes an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like.

  Examples of the steroid hormone in the measurement reagent of the present invention include the aforementioned steroid hormones. Examples of the buffer (I) in the measurement reagent of the present invention include the aforementioned 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 that enables measurement of the steroid hormone of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned aqueous solution. When dissolved in a medium, the content is usually 0.001 to 2.0 moL / L, and preferably 0.005 to 1.0 moL / L.

  Examples of the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant in the measurement reagent of the present invention include, for example, the above-described nonionic surfactant, anionic surfactant, and amphoteric surfactant. Surfactant is mentioned, respectively. The content of each of the nonionic surfactant, the anionic surfactant and the amphoteric surfactant in the measurement reagent of the present invention is a content enabling measurement of the steroid hormone of the present invention. If it is, there will be no restriction | limiting in particular, Usually, it is the content which will be 0.005-3% in the state melt | dissolved in the above-mentioned aqueous medium or the above-mentioned aqueous medium, and the content which will be 0.01-2% is preferable.

  In the measuring 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.

  Examples of the labeled competitive substance, anti-steroid hormone antibody, labeled anti-steroid hormone antibody, and competitive substance in the measurement reagent of the present invention include the aforementioned labeled competitive substance, anti-steroid hormone antibody, labeled anti-steroid hormone antibody, and competition Each substance is listed.

  In the measurement reagent of the present invention, instead of an insoluble carrier on which an antisteroid hormone antibody is immobilized, an antisteroid hormone antibody to which one of a pair of affinity substances (A) is bound and a set of affinity substances 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 antisteroid hormone antibody is immobilized is generated in the reaction solution of the reaction between the specimen or the labeled competitor and the antisteroid hormone antibody. Examples of the combination of one set of affinity substances, that is, the combination of A and a include the aforementioned combinations.

  In the measurement reagent of the present invention, instead of the insoluble carrier on which the competitive substance is immobilized, the competitive substance bound with one of the pair of affinity substances (B) and the other of the pair of affinity substances. A combination with an insoluble carrier to which (b) is bound can also be used. In this case, the insoluble carrier on which the competitor is immobilized is produced in the reaction solution of the reaction between the specimen or the competitor and the labeled antisteroid hormone antibody. Examples of the combination of a pair of affinity substances, that is, the combination of B and b include, for example, the combinations described above.

  The measurement reagent of the present invention may contain a sample pretreatment reagent for pretreating the sample. Examples of the pretreatment include dilution, solubilization, and denaturation. Examples of the specimen pretreatment reagent include the aforementioned aqueous medium. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like. The specimen pretreatment reagent may be liquid or lyophilized. When the specimen pretreatment reagent in a lyophilized state is used, it can be dissolved in an aqueous medium in advance and used for specimen pretreatment. As an aqueous medium, the above-mentioned aqueous medium etc. are mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like.

(Measurement kit)
The measuring reagent of the present invention can be in 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 steroid hormones of the present invention.

  The kit for measuring steroid hormones in a sample of the present invention comprises a first reagent containing an antisteroid hormone antibody and a second reagent containing a labeled competitor, a nonionic surfactant, an anionic interface A kit in which at least one surfactant selected from the group consisting of an active agent and an amphoteric surfactant and the buffer (I) are included in either or both of the first reagent and the second reagent. is there.

  In the measurement kit of the present invention, the antisteroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

  The kit for measuring steroid hormone in a sample of the present invention contains a first reagent containing a labeled antisteroid hormone antibody and a second reagent containing a competitor, and contains a nonionic surfactant, an anion. Each of the first reagent, the second reagent, or both of at least one surfactant selected from the group consisting of an ionic surfactant and an amphoteric surfactant, and the buffer (I) is included. It is a kit.

  In the measurement kit of the present invention, the competitive substance may or may not be immobilized on the insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

  The measurement kit of the present invention may be lyophilized or liquid. When using a measurement kit in a lyophilized state, the measurement kit is dissolved in an aqueous medium before measurement and used for measurement. In the liquid measurement kit, at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, a buffer (I), a labeling agent The competitor, labeled antisteroid hormone antibody, competitor, and antisteroid hormone antibody are dissolved in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like. The liquid measurement kit includes an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like.

  Examples of the steroid hormone in the measurement kit of the present invention include the aforementioned steroid hormones. Examples of the buffer (I) in the measurement kit of the present invention include the aforementioned buffer (I). As the content of the buffer (I) in the first reagent containing the antisteroid hormone antibody and the second reagent containing the labeled competitor in the measurement kit of the present invention, the steroid hormone of the present invention can be measured. There is no particular limitation as long as it is a content to be, usually a content that becomes 0.001 to 2.0 moL / L in a state dissolved in the above-mentioned aqueous medium or in an aqueous medium, and a content that becomes 0.005 to 1.0 moL / L Is preferred. The content of the buffer (I) in the first reagent containing the labeled antisteroid hormone antibody and the second reagent containing the competitor in the measurement kit of the present invention is the measurement of the steroid hormone of the present invention. The content is 0.001 to 2.0 moL / L in the above-mentioned aqueous medium or dissolved in the aqueous medium, and 0.005 to 1.0 moL / L. Is preferred.

  Examples of the nonionic surfactant, the anionic surfactant, and the amphoteric surfactant in the measurement kit of the present invention include the aforementioned nonionic surfactant, anionic surfactant, and amphoteric surfactant. Surfactant is mentioned, respectively. Nonionic surfactant, anionic surfactant, and amphoteric surfactant in the first reagent containing the antisteroid hormone antibody and the second reagent containing the labeled competitor in the measurement kit of the present invention The content of each surfactant in the agent is not particularly limited as long as it enables the measurement of the steroid hormone of the present invention, and is usually in the above-mentioned aqueous medium or in a state dissolved in the aqueous medium. The content is 0.02 to 10%, and the content is preferably 0.04 to 5%. Further, the nonionic surfactant, the anionic surfactant, and the amphoteric in the first reagent containing the labeled antisteroid hormone antibody and the second reagent containing the competitive substance of the measurement kit of the present invention The content of each surfactant in the surfactant is not particularly limited as long as it is a content that enables measurement of the steroid hormone of the present invention, and is usually dissolved in the aforementioned aqueous medium or in the aqueous medium. The content is 0.02 to 10% in the state, and the content that is 0.04 to 5% is preferable.

  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.

  Examples of the labeled competitor, antisteroid hormone antibody, labeled antisteroid hormone antibody, and competitor in the measurement kit of the present invention include the aforementioned labeled competitor, antisteroid hormone antibody, labeled antisteroid hormone antibody, and competitor. Each substance is listed.

In the measurement kit of the present invention, instead of an insoluble carrier on which an antisteroid hormone antibody is immobilized, an antisteroid hormone antibody to which one of a pair of affinity substances (A) is bound and a set of affinity substances 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 antisteroid hormone antibody is immobilized is generated in the reaction solution of the reaction between the specimen or the labeled competitor and the antisteroid hormone antibody. In the measurement kit of the present invention, a combination of an antisteroid hormone antibody bound to one of a pair of affinity substances (A) and an insoluble carrier bound to the other of the pair of affinity substances (a) is used. In this case, the anti-steroid hormone antibody bound to one of the pair of affinity substances (A) and the insoluble carrier bound to the other of the pair of affinity substances (a) may be contained in the same reagent. Although it may be contained in separate reagents, it is preferably contained in separate reagents. 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 set of affinity substances (a) is bound are contained in separate reagents. In this case, a first reagent containing an antisteroid hormone antibody bound to one of a set of affinity substances (A), a second reagent containing a labeled competitor, and the other set of affinity substances (a) A kit containing a third reagent containing an insoluble carrier to which is bound is also included in the kit of the present invention. Examples of the combination of one set of affinity substances, that is, the combination of A and a include the aforementioned combinations. When the insoluble carrier on which the antisteroid hormone antibody is immobilized is produced in the reaction solution of the antigen-antibody reaction, examples of the measurement kit of the present invention include the following kits.
・ Kit 1
A first reagent containing an anti-steroid hormone antibody to which one of a set of affinity substances (A) is bound, a labeled competitor, and an insoluble carrier to which the other of the set of affinity substances (a) 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) Each kit is included 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 labeled competitor, and the other set of affinity substances (a) are bound And at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, and a buffering agent. A kit in which each of (I) is contained in at least one 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 competitive substance is immobilized, the competitive substance bound with one of the pair of affinity substances (B) and the other of the pair of affinity substances (b A combination with an insoluble carrier to which) is bound can also be used. In this case, the insoluble carrier on which the competitor is immobilized is produced in the reaction solution of the reaction between the specimen or the competitor and the labeled antisteroid hormone antibody. In the measurement kit of the present invention, when a combination of a competitive substance bound with one of a pair of affinity substances (B) and an insoluble carrier bound with the other of the pair of affinity substances (b) is used, A competing substance to which one of a pair of affinity substances (B) is bound and an insoluble carrier to which the other pair of affinity substances (b) are bound are contained in the same reagent, but are separated from each other. Although it may be included, it is preferable to be included in a separate reagent. In the case of a kit in which a competing substance to which one of a pair of affinity substances (B) is bound and an insoluble carrier to which the other of the pair of affinity substances (b) is bound are contained in separate reagents, A first reagent containing a labeled antisteroid hormone antibody, a second reagent containing a competitor to which one of a pair of affinity substances (B) is bound, and the other of the pair of affinity substances (b) are bound A kit containing the third reagent containing the insoluble carrier is also included in the kit of the present invention. Examples of the combination of a pair of affinity substances, that is, the combination of B and b include, for example, the combinations described above. When the insoluble carrier on which the competitive substance is immobilized is produced in the reaction solution of the antigen-antibody reaction, examples of the measurement kit of the present invention include the following kits.
・ Kit 3
A first reagent comprising an insoluble carrier to which a labeled antisteroid hormone antibody and the other pair of affinity substances (b) are bound, and a competitor to which one of the pair 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) Each kit is included 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 competitor to which one of a pair of affinity substances (B) is bound, and the other of the pair of affinity substances (b) are bound And at least one surfactant selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant, and a buffering agent. A kit in which each of (I) is contained in at least one 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 the sample may be contained. Examples of the pretreatment include dilution, solubilization, and denaturation. Examples of the specimen pretreatment reagent include the aforementioned aqueous medium. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like. The specimen pretreatment reagent may be liquid or lyophilized. When the specimen pretreatment reagent in a lyophilized state is used, it can be dissolved in an aqueous medium in advance and used for specimen pretreatment. As an aqueous medium, the above-mentioned aqueous medium etc. are mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, proteins, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit the scope of the present invention at all.

[Reference Example] Preparation of anti-aldosterone antibody (1) Preparation of immunogen
ALDOSTERONE 3-CMO (Aldosterone 3-Carboxymethyloxime; made by STERALOIDS) dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 100 mg / mL ALDOSTERONE 3-CMO in DMSO solution (25 μL) , 10 mg / mL KLH (Keyhole limpet hemocyanin; manufactured by Thermo Fisher Scientific) in PBS (2 mL), 100 mg / mL EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; DMSO solution (50 μL) of Thermo Fisher Scientific] and DMSO solution (10 μL) of 100 mg / mL NHS (N-hydroxysuccinimide; manufactured by Thermo Fisher Scientific) were added and mixed. The reaction was allowed to proceed at 16 ° C. for 16 hours to bind ALDOSTERONE 3-CMO and KLH to prepare a KLH-bound ALDOSTERONE 3-CMO solution. For the prepared KLH-bound ALDOSTERONE 3-CMO solution, replace the solvent using a PD-10 column (manufactured by GE Healthcare Japan) equilibrated with PBS solution to prepare a PBS solution of KLH-bound ALDOSTERONE 3-CMO. did. The obtained KLH-conjugated ALDOSTERONE 3-CMO in PBS was used as an immunogen.

(2) Preparation of anti-aldosterone polyclonal antibody The immunogen containing 0.5 mg of KLH-conjugated ALDOSTERONE 3-CMO prepared in (1) above was mixed with an adjuvant to make an emulsion, and the resulting KLH-conjugated ALDOSTERONE 3-CMO emulsion was measured. Four times, a rabbit (NZW species) was injected subcutaneously. For the first immunization, Freund's Complete Adjuvant (Sigma Aldrich) is used as an adjuvant. For the second to fourth immunizations, Freund's Incomplete Adjuvant (Sigma Aldrich) is used as an adjuvant at intervals of 2 weeks. Blood was collected after 4 immunizations in total, and the antibody titer in the serum was evaluated by ELISA. After confirming that the antibody titer in the serum increased, Freund's Incomplete Adjuvant (manufactured by Sigma Aldrich) was further used as an adjuvant, and immunization was performed twice at intervals of 2 weeks. 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.

  Aldosterone measurement kits A, B, C, D, and E comprising the following streptavidin-coupled magnetic particle suspension, biotin-conjugated anti-aldosterone antibody solution, alkaline phosphatase labeled competitor solution, and aldosterone standard solution were prepared.

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

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

<Biotin-conjugated 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) are mixed and reacted at 37 ° C. for 1 hour. The mixture after the reaction is separated by Sephadex G -50 column (manufactured by GE Health Science Japan) was used to remove unreacted NHS-biotin, and a biotin-conjugated anti-aldosterone polyclonal antibody was prepared. A biotin-conjugated anti-aldosterone antibody solution having the following composition was prepared using the obtained biotin-conjugated anti-aldosterone polyclonal antibody.

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

<Alkaline phosphatase labeling competitor 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 (manufactured by Thermo Fisher Scientific), reacted at 25 ° C. for 15 minutes, and further reacted with alkaline phosphatase (Roche Diagnostics) at 4 ° C. for 16 hours. Reacted. The reaction mixture was applied 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 labeling competitor, an alkaline phosphatase labeling competitor solution having the following composition was prepared.

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

<Aldosterone standard solution>
Aldosterone (manufactured by Sigma-Aldrich) was diluted with delipidated plasma [Human Plasma, Defibrinated, Delipidized double charcoal stripped (Golden West Biologicals)], and the aldosterone concentration was 0, 16, 40, 80, An aldosterone solution with each concentration of 160, 400, 1600 pg / mL was used as an aldosterone standard solution for preparing a calibration curve.

  The same kind of buffer was used in the streptavidin-conjugated magnetic particle suspension, the biotin-conjugated anti-aldosterone antibody solution, and the alkaline phosphatase-labeled competitor solution.

[Comparative Example 1]
Using a similar method except that phosphate buffer, Tris buffer or citrate buffer is used instead of buffer (I) of Example 1, a suspension of streptavidin-conjugated magnetic particles, biotin-conjugated anti-aldosterone is used. A kit a comprising an antibody solution and an alkaline phosphatase-labeled competitor solution prepared from each streptavidin-conjugated magnetic particle suspension, a biotin-conjugated anti-aldosterone antibody solution, an alkaline phosphatase-labeled competitor solution, and the aldosterone standard solution of Example 1 , B and c were prepared.

(1) Preparation of calibration curve Add each aldosterone standard solution of Example 1 (10 μL) and the biotin-conjugated anti-aldosterone antibody solution of Example 1 (50 μL) to the reaction vessel, stir, and continue at 37 ° C. for 18 minutes. After the reaction, the streptavidin-coupled magnetic particle suspension of Example 1 (30 μL) and the alkaline phosphatase labeled competitor solution (30 μL) were added to the reaction solution and stirred, and reacted at 37 ° C. for 8 minutes. It was. After collecting the magnetic particles by magnetic force and removing the reaction solution other than the magnetic particles, a washing solution [50 mmol / L MOPS buffer (pH 7.35) containing 0.1% Tween 20] is added to the reaction vessel. Was washed. After collecting the magnetic particles by magnetic force and removing the solution other than the magnetic particles, a cleaning solution was added to the reaction vessel to wash the magnetic particles. This series of magnetic collection, removal of solutions other than magnetic particles, and washing of magnetic particles with a washing solution was performed five times, and then 9- (4-chlorophenylthiophosphoryloxymethylidene) -10-methylacridan / disodium A luminescent substrate solution (70 μL) containing salt as a main component was added and stirred, and the generated luminescence (RLU) was measured to prepare a calibration curve 1 representing the relationship between the aldosterone concentration and the luminescence (RLU).

  Instead of each aldosterone standard solution, biotin-conjugated anti-aldosterone antibody solution, streptavidin-conjugated magnetic particle suspension, and alkaline phosphatase-labeled competitor solution in the kit of Example 1, each aldosterone standard solution of Comparative Example 1, biotin binding A calibration curve 2 representing the relationship between aldosterone concentration and luminescence level (RLU) was obtained in the same manner as described above, except that an anti-aldosterone antibody solution, a streptavidin-binding magnetic particle suspension, and an alkaline phosphatase-labeled competitor solution were used. Created.

(2) Determination of aldosterone concentration in specimen-correlation test 13 serum or plasma specimens collected from primary aldosteronism patients and healthy persons instead of the aldosterone standard solutions of Example 1 and Comparative Example 1 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 specimen was determined from the luminescence (RLU) obtained for each specimen and the calibration curve prepared in (1). When determining the aldosterone concentration using the kit of Example 1, the calibration curve 1 is used as the calibration curve, and when determining the aldosterone concentration using the kit of Comparative Example 1, the calibration curve is used as the calibration curve. 2 was used.

In addition, using a commercially available aldosterone measurement kit “SPAC-S aldosterone kit” (manufactured by Fujirebio Inc.), measurement was performed according to the measurement procedure described in the package insert of the kit, It was determined.
One of the methods for determining whether or not accurate measurement is possible is a correlation test. The test involves measuring a plurality of identical specimens using existing in vitro diagnostic drugs and an evaluation reagent to be compared with the in vitro diagnostic drugs, and measuring values obtained by measurement using the respective reagents. This is a test for statistically calculating the correlation equation (Y = aX + b) and the correlation coefficient (r) and evaluating 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 is the same as the measured value in the evaluation reagent. This means that accurate 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 “SPAC-S aldosterone kit” (manufactured by Fujirebio), and the correlation coefficient Is shown in Table 1.

  As is apparent 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 using the buffer (I), The slopes of the correlation equations with the aldosterone measurement using the “S aldosterone kit” (Fujirebio Inc.) are 0.80, 0.81, and 1.37, respectively. The measurement of the aldosterone used, that is, the measurement of the aldosterone of the present invention using the buffer (I) and the nonionic surfactant is between the measurement of the aldosterone using the “SPAK-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 aldosterone measurement method of the present invention using the buffer (I) and the nonionic surfactant is a method that can accurately measure aldosterone.

In the measurement of aldosterone using the kits A to E of Example 1 and the kits a to c of Comparative Example 1, the cross-reactivity with respect to each cross-reactive substance of corticosterone, cortisol and cortisone was evaluated. Corticosterone, cortisol and cortisone are all steroid hormones similar in structure to aldosterone.
Each cross-reactive substance was prepared by diluting with degreased plasma [Human Plasma, Defibrinated, Delipidized double charcoal stripped (Golden West Biologicals)], 10 to 250 μg / mL (= 1 × 10 ^{7 to} 2.5). × 10 ⁸ pg / mL) each cross-reactive substance solution was used as a specimen, and measurement was performed according to the method of Example 2 (2). The cross-rate for each cross-reactive substance was determined by the following equation. . The results are shown in Table 2.

  The crossing rate is an index indicating the rate at which cross-reactive substances that should not be measured should be measured. Therefore, the lower the crossing rate, the more specifically aldosterone can be detected and the more accurate it can be measured. It shows that.

  As is apparent from Table 2, the kit was compared with the measurement of aldosterone using each kit of 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 kit of A to E, that is, 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 aldosterone measurement method of the present invention using the buffer (I) and the nonionic surfactant is a method that can accurately measure aldosterone.

  Aldosterone measurement kits F to Q comprising the following streptavidin-coupled magnetic particle suspension, biotin-conjugated anti-aldosterone antibody solution, alkaline phosphatase labeled competitor solution, and aldosterone standard solution were prepared.

<Streptavidin-bound magnetic particle suspension>
Streptavidin-bonded magnetic particle suspension (pH 6.5) having the following composition using commercially available magnetic particles (Dynabeads M-280 Streptavidin: manufactured by Thermo Fisher Scientific Co.) bound with streptavidin as magnetic particles. Was prepared.

BES 125 mmol / L
Streptavidin-bound magnetic particles 0.7 mg / mL
BSA 0.1%
Surfactant (See Table 3)

<Biotin-conjugated anti-aldosterone antibody solution>
A biotin-conjugated anti-aldosterone antibody solution (pH 6.5) having the following composition using the anti-aldosterone polyclonal antibody obtained in Reference Example and the biotin-conjugated anti-aldosterone antibody obtained in the same manner as in Example 1. Was prepared.

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

<Alkaline phosphatase labeling competitor solution>
Using an alkaline phosphatase labeling competitor prepared by the same method as in Example 1, an alkaline phosphatase labeling competitor solution (pH 7.5) having the following composition was prepared.

TES 20 mmol / L
Alkaline phosphatase labeling competitor 1.5μg / mL
BSA 0.1%
Surfactant (See Table 3)
Magnesium chloride hexahydrate 30 mmol / L

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

  The surfactant in the suspension of streptavidin-coupled magnetic particles and the surfactant in the alkaline phosphatase-labeled competitive substance solution were the same type and concentration.

[Comparative Example 2]
Instead of the streptavidin-binding magnetic particle suspension and alkaline phosphatase-labeled competitor solution in the kit of Example 4, the surfactant-free streptavidin-binding magnetic particle suspension and alkaline phosphatase-labeled competitor A kit comprising a solution containing a surfactant-free streptavidin-conjugated magnetic particle suspension, a biotin-conjugated anti-aldosterone antibody solution, an alkaline phosphatase-labeled competitor solution containing no surfactant, and the aldosterone standard solution of Example 4 d was produced.

(1) Preparation of calibration curve Add each aldosterone standard solution of Example 4 (10 μL) and the biotin-conjugated anti-aldosterone antibody solution of Example 4 (50 μL) to the reaction vessel, and stir at 37 ° C. for 18 minutes. After the reaction, the streptavidin-coupled magnetic particle suspension of Example 4 (30 μL) and the alkaline phosphatase-labeled competitor solution (30 μL) were added to the reaction solution and stirred, and reacted at 37 ° C. for 8 minutes. It was. After collecting the magnetic particles by magnetic force and removing the reaction solution other than the magnetic particles, a washing solution [50 mmol / L MOPS buffer (pH 7.35) containing 0.1% Tween 20] is added to the reaction vessel. Was washed. After collecting the magnetic particles by magnetic force and removing the solution other than the magnetic particles, a cleaning solution was added to the reaction vessel to wash the magnetic particles. This series of magnetic collection, removal of solutions other than magnetic particles, and washing of magnetic particles with a washing solution was performed five times, and then 9- (4-chlorophenylthiophosphoryloxymethylidene) -10-methylacridan / disodium A luminescent substrate solution (70 μL) containing salt as a main component was added and stirred, and the generated luminescence (RLU) was measured, and a calibration curve 3 representing the relationship between aldosterone concentration and luminescence (RLU) was prepared.

  Instead 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 in the kit of Example 4, each aldosterone of Comparative Example 2 Aldosterone concentration and luminescence level in the same manner as described above except that each solution or suspension of standard solution, biotin-conjugated anti-aldosterone antibody solution, streptavidin-conjugated magnetic particle suspension, and alkaline phosphatase labeled competitor solution is used. A calibration curve 4 representing the relationship with (RLU) was created.

(2) Determination of aldosterone concentration in specimen-correlation test 13 serum or plasma specimens collected from primary aldosteronism patients and healthy persons instead of the aldosterone standard solutions of Example 4 and Comparative Example 2 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 specimen was determined from the luminescence (RLU) obtained for each specimen and the calibration curve prepared in (1). When determining the aldosterone concentration using the kit of Example 4, the calibration curve 3 is used as the calibration curve, and when determining the aldosterone concentration using the kit of Comparative Example 2, the calibration curve is used as the calibration curve. 4 was used.

In addition, using a commercially available aldosterone measurement kit “SPAC-S aldosterone kit” (manufactured by Fujirebio Inc.), measurement was performed according to the measurement procedure described in the package insert of the kit, It was determined.
Measurement of aldosterone using the streptavidin-conjugated magnetic particle suspension, biotin-conjugated anti-aldosterone antibody solution, and alkaline phosphatase-labeled competitor solution prepared in Example 4 and Comparative Example 2, and “SPAC-S aldosterone kit” (Fuji Table 3 shows the slope of the correlation equation and the correlation coefficient between the aldosterone measurement using Rebio).

  As apparent from Table 3, measurement of aldosterone using the kit of kit d, that is, using only buffer (I), nonionic surfactant, anionic surfactant, and amphoteric surfactant The measurement of aldosterone without using any of the above surfactants has a slope of 1.30 in the correlation equation with the measurement of aldosterone using “SPAC-S aldosterone kit” (manufactured by Fujirebio Inc.), and accurate measurement cannot be performed. In contrast, measurement of aldosterone using each kit of kits F to Q, that is, comprising buffer (I) and a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant The measurement of aldosterone of the present invention using at least one selected from the group is performed using the “SPAC-S aldosterone kit” (Fujirebio Co., Ltd.). The slope of the correlation equation between the measurement of Teron a is 0.86 to 1.21, close to almost 1.0, and the correlation coefficient was 0.97 to 1.00, it was close to about 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, It proved to be a method that can accurately measure aldosterone.

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

Each cross-reactive substance was prepared by diluting with degreased plasma [Human Plasma, Defibrinated, Delipidized double charcoal stripped (Golden West Biologicals)], 10 to 250 μg / mL (= 1 × 10 ^{7 to} 2.5). X10 ⁸ pg / mL) is used as a specimen, and measurement is performed according to the method of Example 5 (2). By the same formula as Example 3, the crossover rate for each cross-reactive substance is measured. It was determined. The results are shown in Table 4.

  As apparent from Table 4, measurement of aldosterone using kit d, that is, using only buffer (I), any of nonionic surfactants, anionic surfactants, and amphoteric surfactants Compared with measurement of aldosterone without using surfactant, measurement of aldosterone using kits F to Q, that is, buffer (I), nonionic surfactant, anionic surfactant In the measurement of aldosterone using at least one surfactant selected from the group consisting of amphoteric surfactants, the 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 measurement method was found to be a method capable of accurately measuring aldosterone.

  The present invention provides a method for measuring steroid hormones in a specimen, a reagent for measurement, and a kit for measurement, which are useful for clinical diagnosis.

Claims (23)

A specimen, an antisteroid hormone antibody and a labeled competitor are represented by the general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), 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 the antisteroid hormone antibody and the labeled competitor, A method for measuring a steroid hormone in a specimen, which comprises measuring a label in an immune complex. The measurement method according to claim 1, wherein after the sample and the antisteroid hormone antibody are reacted, a labeled competitor is added to the reaction solution. The measurement method according to claim 1 or 2, wherein the antisteroid hormone antibody is immobilized on an insoluble carrier. A sample, a labeled antisteroid hormone antibody, and a competitor are represented by the general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a nonionic surfactant, an anionic surfactant, and Reacting in an aqueous medium containing at least one surfactant selected from the group consisting of amphoteric surfactants to generate an immune complex between the labeled antisteroid hormone antibody and the competitor, A method for measuring a steroid hormone in a specimen, which comprises measuring a label in an immune complex. The measurement method according to claim 4, wherein after the sample and the labeled antisteroid hormone antibody are reacted, a competitive substance is added to the reaction solution of the reaction. The measuring method according to claim 4 or 5, wherein the competitive substance is immobilized on an insoluble carrier. Buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N- Bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES), and bis (2-hydroxyethyl) iminotris (hydroxymethyl) The measurement method according to claim 1, which is a buffer selected from the group consisting of methane (Bis-Tris). The measurement method according to any one of claims 1 to 7, wherein the steroid hormone is a mineral corticoid. The measuring method according to claim 8, wherein the mineral corticoid is aldosterone. At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, general formula (I)
(Wherein, X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5) in a sample containing a buffer having a structure represented by: Reagent for measuring steroid hormones. The reagent according to claim 10, wherein the antisteroid hormone antibody is immobilized on an insoluble carrier. At least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants, general formula (I)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), a buffering agent having a structure represented by: a labeled antisteroid hormone antibody, and a competitor, Reagent for measuring steroid hormones. The reagent according to claim 12, wherein the competitive substance is immobilized on an insoluble carrier. Buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N- Bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES), and bis (2-hydroxyethyl) iminotris (hydroxymethyl) The reagent according to any one of claims 10 to 13, which is a buffer selected from the group consisting of methane (Bis-Tris). The reagent according to any one of claims 10 to 14, wherein the steroid hormone is a mineralocorticoid. The reagent according to claim 15, wherein the mineralocorticoid is aldosterone. A first reagent containing an antisteroid hormone antibody and a second reagent containing a labeled competitor are selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. At least one surfactant selected from general formula (I)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), each of the buffering agents is included in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in a specimen. The kit according to claim 17, wherein the antisteroid hormone antibody is immobilized on an insoluble carrier. A first reagent containing a labeled antisteroid hormone antibody and a second reagent containing a competitor are selected from the group consisting of a nonionic surfactant, an anionic surfactant, and an amphoteric surfactant. At least one surfactant selected from general formula (I)
(Wherein X represents a hydroxyl group or a sulfo group, and n represents an integer of 2 to 5), each of the buffering agents is included in either or both of the first reagent and the second reagent. A kit for measuring steroid hormones in a specimen. The kit according to claim 19, wherein the competitive substance is immobilized on an insoluble carrier. Buffers are 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 2- [N, N- Bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES), N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES), and bis (2-hydroxyethyl) iminotris (hydroxymethyl) The kit according to any one of claims 17 to 20, which is a buffer selected from the group consisting of methane (Bis-Tris). The kit according to any one of claims 17 to 21, wherein the steroid hormone is a mineralocorticoid. The kit according to claim 22, wherein the mineralocorticoid is aldosterone.