JP7579190B2 - Method for stabilizing a kit for measuring steroid hormones in a sample by competitive method - Google Patents

Description

The present invention relates to a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method, a kit for measuring steroid hormones in a sample by a competitive method, a method for stabilizing a labeled steroid hormone, and a reagent containing a labeled steroid hormone.
This application claims priority based on Japanese Patent Application No. 2020-061065, filed on March 30, 2020, the contents of which are incorporated herein by reference.

Steroid hormones are hormones with a steroid skeleton and a molecular weight of 300-400. Based on their functions, they are classified into sex hormones, glucocorticoids, mineralocorticoids, etc. For clinical research on steroid hormones, methods for measuring the amount of steroid hormones in the body have been studied, and gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), immunoassays, etc. have been developed. Although GC-MS and LC-MS have high analytical capabilities, there are problems such as the high cost of the analytical equipment used for these analyses and the need for complicated operations such as concentration and extraction. On the other hand, immunoassays are often used in clinical research because they can measure steroid hormones easily and in a short time.

Aldosterone is a mineralocorticoid with a molecular weight of approximately 360 that is secreted from the zona glomerulosa, which is part of the outer layer of the adrenal cortex, and its secretion is mainly dependent on the renin-angiotensin-aldosterone system. Aldosterone is a hormone that is extremely important for maintaining electrolyte homeostasis, circulating blood volume, and blood pressure, and acts on the mineralocorticoid receptors in the distal renal tubules to promote the reabsorption of water as well as sodium, thereby increasing blood pressure.

Aldosterone is used in the differential diagnosis of primary aldosteronism, Bartter's syndrome, Liddle's syndrome, hydroxylase deficiencies such as 17α-hydroxylase deficiency and 11β-hydroxylase deficiency in the adrenal glands, and selective hypoaldosteronism. In addition, the Japan Endocrine Society Guidelines and the Japan Society of Hypertension Guidelines recommend the measurement of the aldosterone to renin ratio (ARR), which is the ratio of aldosterone concentration (PAC) to renin activity (PRA) or renin concentration (Active Renin Concentration (ARC)), as a screening method for primary aldosteronism.

Methods for measuring aldosterone include the LC-MS method and immunological measurement methods, and immunological measurement methods include the radioimmunoassay method (RIA method) and the enzyme immunoassay method (EIA method) (see Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2-057976 JP 2017-142241 A

Known immunoassay methods include the sandwich method and the competitive method. Usually, the sandwich method is used when measuring macromolecules such as proteins, and the competitive method is often used when measuring low molecular weight compounds such as steroid hormones.

The sandwich method is a method for measuring the component to be measured in a sample by, for example, reacting a component to be measured in a sample with a first antibody immobilized on a solid phase and a labeled second antibody in an aqueous medium to generate an immune complex consisting of the component to be measured, the first antibody immobilized on the solid phase, and the labeled second antibody, and then measuring the label in the generated immune complex. In a measurement kit using the sandwich method, the stability of the kit during long-term storage can be improved by increasing the amount of the first antibody and the amount of the labeled second antibody.

On the other hand, in the competitive method, for example, a component to be measured in a sample, an antibody immobilized on a solid phase, and a labeled competitor are reacted in an aqueous medium to generate an immune complex 1 consisting of the component to be measured and the antibody immobilized on the solid phase, and an immune complex 2 consisting of the labeled competitor and the antibody immobilized on the solid phase, and then the label in the generated immune complex 2 is measured. At this time, the component to be measured and the competitor competitively bind to the antibody immobilized on the solid phase, so that the amounts of immune complexes 1 and 2 generated are inversely correlated. In this way, the amount of immune complex 1 generated, i.e., the amount of the component to be measured in the sample, can be measured from the measurement result of the label in the immune complex 2.

As with measurement kits using the sandwich method, it is necessary to improve the stability during long-term storage of measurement kits using the competitive method. However, for example, when a large amount of labeled competing substance is present in the immune reaction, immune complex 2 is preferentially produced and the amount of immune complex 1 produced is very small, making it impossible to accurately measure the amount of the component to be measured in the sample. Therefore, in measurement kits using the competitive method, it is difficult to improve the stability during long-term storage by increasing the amount of labeled competing substance.

In order to solve the above problems, the present invention aims to provide a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method; a kit for measuring steroid hormones in a sample by a competitive method that has excellent stability during long-term storage; a method for stabilizing a labeled steroid hormone; and a labeled steroid hormone-containing reagent that has excellent stability during long-term storage.

As a result of intensive research to solve this problem, the present inventors have found that, in a kit for measuring steroid hormones in a sample by a competitive method, the kit comprises a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone, by allowing the labeled steroid hormone and bovine serum albumin to coexist in an aqueous medium in the second reagent, the stability of the kit during long-term storage is improved, and thus the present invention has been completed. That is, the present invention relates to the following [1] to [28].

[1] A method for stabilizing a kit for measuring a steroid hormone in a sample by a competitive method, the kit comprising a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone, the method comprising:
A method for stabilizing said steroid hormone, comprising the step of causing said labeled steroid hormone to coexist with 10 g/L or more of bovine serum albumin in an aqueous medium in said second reagent.
[2] The stabilization method according to [1], wherein the concentration of the bovine serum albumin is 20 to 30 g/L.
[3] The stabilization method according to [1] or [2], wherein the pH of the aqueous medium is 7 to 8.
[4] The stabilization method described in any one of [1] to [3], wherein the labeled steroid hormone is an enzyme-labeled steroid hormone.
[5] The stabilization method described in [4], wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase.
[6] The stabilization method described in any one of [1] to [5], wherein the steroid hormone is a mineralocorticoid.
[7] The stabilization method described in [6], wherein the mineralocorticoid is aldosterone.

[8] a first reagent comprising an anti-steroid hormone antibody;
a second reagent comprising an aqueous medium, a labeled steroid hormone, and bovine serum albumin at 10 g/L or more;
A kit for measuring a steroid hormone in a sample by a competitive method, comprising:
[9] The kit according to [8], wherein the concentration of the bovine serum albumin is 20 to 30 g/L.
[10] The kit according to [8] or [9], wherein the aqueous medium has a pH of 7 to 8.
[11] The kit according to any one of [8] to [10], wherein the labeled steroid hormone is an enzyme-labeled steroid hormone.
[12] The kit according to [11], wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase.
[13] The kit according to any one of [9] to [12], wherein the steroid hormone is a mineralocorticoid.
[14] The kit described in [13], wherein the mineralocorticoid is aldosterone.

[15] A method for stabilizing a labeled steroid hormone, which comprises allowing the labeled steroid hormone to coexist with 10 g/L or more of bovine serum albumin in an aqueous medium.
[16] The stabilization method according to [15], wherein the concentration of the bovine serum albumin is 20 to 30 g/L.
[17] The stabilization method according to [15] or [16], wherein the pH of the aqueous medium is 7 to 8.
[18] The stabilization method according to any one of [15] to [17], wherein the labeled steroid hormone is an enzyme-labeled steroid hormone.
[19] The stabilization method described in [18], wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase.
[20] The stabilization method described in any one of [15] to [19], wherein the steroid hormone is a mineralocorticoid.
[21] The stabilization method described in [20], wherein the mineralocorticoid is aldosterone.

[22] A method for measuring a steroid hormone in a sample by a competitive method, which comprises reacting a steroid hormone in the sample with an anti-steroid hormone antibody and a labeled steroid hormone to form an immune complex between the anti-steroid hormone antibody and the labeled steroid hormone, and measuring the label in the immune complex thus formed.
A labeled steroid hormone-containing reagent for use in
A labeled steroid hormone-containing reagent, comprising a labeled steroid hormone and bovine serum albumin at a concentration of 10 g/L or more in an aqueous medium.
[23] The reagent according to [22], wherein the concentration of the bovine serum albumin is 20 to 30 g/L.
[24] The reagent according to [22] or [23], wherein the aqueous medium has a pH of 7 to 8.
[25] The reagent according to any one of [22] to [24], wherein the labeled steroid hormone is an enzyme-labeled steroid hormone.
[26] The reagent described in [25], wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase.
[27] The reagent according to any one of [22] to [26], wherein the steroid hormone is a mineralocorticoid.
[28] The reagent described in [27], wherein the mineralocorticoid is aldosterone.

The present invention provides a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method; a kit for measuring steroid hormones in a sample by a competitive method that has excellent stability during long-term storage; a method for stabilizing a labeled steroid hormone; and a labeled steroid hormone-containing reagent that has excellent stability during long-term storage. The invention also provides a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method without reducing the measurement signal.

Hereinafter, the embodiments of the method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method, the kit for measuring steroid hormones in a sample by a competitive method, the method for stabilizing a labeled steroid hormone, and the labeled steroid hormone-containing reagent of the present invention will be described. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and the scope of the disclosure should not be interpreted narrowly by them.
Note that the numerical range indicated using "to" indicates a range including the numerical values before and after "to" as the minimum and maximum values, respectively. Furthermore, when multiple substances corresponding to each component are present in the solution, the amount of each component in the solution means the total amount of the multiple substances present in the reaction or in the reagent, unless otherwise specified.

(1) Method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method The method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method of the embodiment (hereinafter referred to as the method for stabilizing the measurement kit of the embodiment) is a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method, which comprises a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone, characterized in that in the second reagent, the labeled steroid hormone and 10 g/L or more of bovine serum albumin are allowed to coexist in an aqueous medium.

In the stabilization method of the measurement kit of the embodiment, "stable" means that even when the measurement kit of the embodiment is stored for a long period of time and then used in the measurement method described below, the signal derived from the labeling substance in the immune complex 2 generated in step [2] in the measurement method described below is less likely to decrease compared to before storage. For example, in the stabilization method of the measurement kit of the embodiment, even when the measurement kit for steroid hormones in a sample by the competitive method is stored at 5°C for 140 days and then used in the measurement method described below, the signal derived from the labeling substance in the immune complex 2 is 70% or more compared to before storage.
The signal derived from the labeling substance in the immune complex 2 can be measured, for example, by the following method.

[1] A step of reacting a sample not containing steroid hormone with a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone to produce an immune complex 2 consisting of the labeled steroid hormone and the anti-steroid hormone antibody; and [2] a step of measuring the labeled substance in the immune complex 2 produced in step [1].

Before and after long-term storage of the measurement kit of the embodiment, the signal (e.g., luminescence amount) derived from the labeling substance is measured, and the ratio of the signal after storage to the signal before storage is calculated. Here, for example, if the ratio is higher than 70%, the measurement kit of the embodiment can be evaluated as being stable.

The specimen in the stabilization method of the measurement kit of the embodiment is not particularly limited as long as it is a specimen that can be measured using the measurement kit of the embodiment, and examples thereof include whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, amniotic fluid, sweat, pancreatic juice, etc., with whole blood, plasma, serum, urine, etc. being preferred.

The steroid hormone in the stabilization method of the measurement kit of the embodiment is not particularly limited as long as it is a steroid hormone that enables the stabilization method of the measurement kit of the embodiment, and examples thereof include mineralocorticoids, glucocorticoids, sex hormones, etc., and mineralocorticoids are preferred. Examples of mineralocorticoids include aldosterone, fludrocortisone acetate, etc. Examples of glucocorticoids include cortisol, corticosterone, cortisone, etc. Examples of sex hormones include male hormones (androgens), female hormones, etc. Examples of male hormones include testosterone, dehydroepiandrosterone, etc. Examples of female hormones include estradiol, estriol, estrone, progesterone, etc.

In the method for stabilizing a measurement kit according to the embodiment, the kit for measuring a steroid hormone in a sample by a competitive method (hereinafter referred to as the measurement kit according to the embodiment) includes a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone. The kit may also include a third reagent or other reagents, for example, including an insoluble carrier as described below.

The anti-steroid hormone antibody contained in the first reagent is not particularly limited as long as it is an antibody that binds to a steroid hormone and enables the stabilization method of the measurement kit of the embodiment, and either a polyclonal antibody or a monoclonal antibody can be used. Furthermore, in the embodiment, not only full-length antibodies but also antibody fragments can be used. Examples of antibody fragments include antibody fragments from which the Fc portion has been removed, such as Fab obtained by treating an antibody with papain, F(ab') ₂ obtained by treating with pepsin, and Fab' obtained by treating with pepsin and then reducing.

The anti-steroid hormone antibody 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 a steroid hormone as an antigen, but it is also available as a commercially available product, such as a polyclonal antibody against any steroid hormone (manufactured by Abcam).
The concentration of the anti-steroid hormone antibody is not particularly limited as long as it is a concentration that enables the stabilization method of the measurement kit of the embodiment, and is usually 0.1 to 1000 ng/mL, preferably 1 to 100 ng/mL.

The anti-steroid hormone antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized. When the anti-steroid hormone antibody is immobilized on an insoluble carrier, the insoluble carrier on which the anti-steroid hormone antibody is immobilized may be produced in the reaction solution of the antigen-antibody reaction. In this case, the anti-steroid hormone antibody bound to one of a pair of affinity substances (A) and the insoluble carrier bound to the other of the pair of affinity substances (a) are reacted in the reaction solution of the antigen-antibody reaction to produce the insoluble carrier on which the anti-steroid hormone antibody is immobilized in the reaction solution of the antigen-antibody reaction. Examples of the combination of A-a include the following combinations.
- combinations of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
- combination of avidins (avidin, neutravidin, streptavidin, etc.) with biotin;
A combination of the Fc region of an anti-steroid hormone antibody and an antibody that binds to the Fc region.

The insoluble carrier in the embodiment is not particularly limited as long as it is an insoluble carrier capable of immobilizing an anti-steroid hormone antibody and a competing substance and enabling the stabilization method of the measurement kit of the embodiment, and examples thereof include polystyrene plates such as microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, various membranes such as nitrocellulose membranes, synthetic resin test tubes, and the like.
Furthermore, when an insoluble carrier having an anti-steroid hormone antibody immobilized thereon is produced in the reaction solution of the antigen-antibody reaction, the insoluble carrier is contained in the first reagent, the second reagent or another reagent.

The first reagent may contain, as necessary, an aqueous medium, a preservative, a salt, a sugar, a surfactant, a protein, an agent for eliminating interferences, and the like.
The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the stabilization method of the measurement kit of the embodiment, and examples thereof include deionized water, distilled water, buffer solution, etc., with buffer solution being preferred. The pH of the buffer solution is also not particularly limited as long as it enables the stabilization method of the measurement kit of the embodiment, and may be, for example, pH 4 or more, pH 5 or more, pH 6 or more, or pH 7 or more, and may be pH 8 or less or pH 9 or less, with pH 6 to 8 being preferred, and pH 7 to 8 being more preferred.
In addition, in the present disclosure, a numerical range obtained by combining any numerical value selected from the upper limit example and the lower limit example for a certain amount is also considered to be exemplified in the present disclosure. The same applies to other numerical values such as concentration, period, temperature, etc.

Examples of buffering agents used in the preparation of the buffer solution 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), and bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris). , N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).
The concentration of the buffer is not particularly limited as long as it enables the stabilization method of the measurement kit of the embodiment, and is usually 0.001 to 2.0 mol/L, preferably 0.005 to 1.0 mol/L.

Examples of preservatives include sodium azide and antibiotics. Examples of salts include sodium chloride, sodium nitrate, sodium sulfate, sodium carbonate, sodium formate, sodium acetate, potassium chloride, potassium nitrate, potassium sulfate, potassium carbonate, potassium formate, potassium acetate, and ethylenediaminetetraacetic acid (EDTA). Examples of sugars include trehalose and sucrose. Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. Examples of proteins include bovine serum albumin. Examples of interference removers include ascorbic acid oxidase for removing the effects of ascorbic acid.

The labeling substance used in the labeled steroid hormone is not particularly limited as long as it enables the stabilization method of the measurement kit of the embodiment, and examples thereof include enzymes, fluorescent substances, luminescent substances, radioisotopes, biotin, digoxigenin, polypeptides containing tag sequences, metal colloid particles, colored latex particles, etc.

Examples of enzymes include alkaline phosphatase, peroxidase, β-D-galactosidase, luciferase, etc.

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

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

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

Labeling of steroid hormones with labeling substances can be carried out by a reaction that produces a covalent bond between a functional group of the steroid hormone and a functional group of the labeling substance, with or without a linker. Examples of functional groups include carboxyl groups, amino groups, glycidyl groups, sulfhydryl groups, hydroxyl groups, amide groups, imino groups, hydroxysuccinyl ester groups, maleimide groups, and isothiocyanate groups.

When labeling a steroid hormone with a labeling substance, the position in the steroid hormone where the steroid hormone binds to the labeling substance can be appropriately selected depending on the structure of the steroid hormone. For example, when the steroid hormone to be measured is aldosterone and labeled aldosterone is used as the labeled steroid hormone, the labeling substance can be bound to the 3rd, 4th, 6th, 21st, or other positions of aldosterone.

Examples of linking methods that do not involve 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 its derivatives. The condensation reaction between an isothiocyanate group and an amino group is preferable because it does not require other reagents and proceeds simply by mixing under neutral to weakly alkaline conditions.

The linker may be any linker that can bond the functional group of the steroid hormone and the functional group of the labeling substance. A preferred embodiment is, for example, a molecule having a first functional group capable of reacting with the carboxyl group of the steroid hormone and a second functional group capable of reacting with the functional group of the labeling substance in the same molecule, and it is preferable that the first functional group and the second functional group are different. Examples of the functional group of the linker include the above-mentioned functional groups.

Methods for chemically binding a radioisotope include, for example, the methods described in the literature (Antibody Immunoconj. Radiopharm., 3, 60, 1990).

When the labeling substance is an enzyme, avidin, a fluorescent protein, a phycobiliprotein, a polypeptide containing a tag sequence, or the like, it can be produced by preparing an expression vector containing DNA encoding a fusion protein of the labeling substance and an anti-steroid hormone antibody according to known recombinant gene technology (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001), introducing the expression vector into a suitable host, and culturing the host. The DNA encoding the fusion protein can be obtained by cloning the DNAs encoding the antibody and labeling substance by PCR or the like, and linking the respective DNAs by a ligase reaction.

The measurement of the labeled substance in the immune complex between the anti-steroid hormone antibody and the labeled steroid hormone can be appropriately selected depending on the labeled substance used.

When the labeling substance is a color-developing 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 luminescence multiwell plate reader, or the like.
When the labeling substance is a radioisotope, the radioactivity can be measured using 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 the enzyme's substrate with the enzyme and measuring the amount of the product.

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

Examples of substrates for alkaline phosphatase 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[3.3.1.1 ^3.7 ]decane]-4-yl}phenyl phosphate disodium salt (CDP-Star ^™ ), 3-{4-
methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 ^3.7 ]decane]-4-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 peroxidase, peroxidase activity can be measured, for example, by absorbance or fluorescence. Examples of methods for measuring peroxidase activity by absorbance include reacting peroxidase with a combination of its substrates, hydrogen peroxide, and an oxidative color-developing chromogen, and measuring the absorbance of the reaction solution using a spectrophotometer or multiwell plate reader. Examples of oxidative color-developing chromogens include leuco chromogens and oxidative coupling color-developing chromogens.

Leuco chromogens are substances that are converted to dyes by themselves in the presence of peroxidatively active substances such as hydrogen peroxide and peroxidase. Specific examples include tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67), 4,4'-bis(dimethylamino)diphenylamine, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA), and the like.

Oxidative coupling color-developing chromogens are substances that generate dyes by oxidative coupling of two compounds in the presence of hydrogen peroxide and a peroxidatively active substance such as peroxidase. Examples of combinations of two compounds include a combination of a coupler and an aniline (Trinder's reagent) and a combination of a coupler and a phenol.

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

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)-3-methoxyaniline, ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS), etc.

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

One example of a method for measuring peroxidase activity using a fluorescence method is to react peroxidase with a combination of its substrate, hydrogen peroxide, and a fluorescent substance, and measure the intensity of the fluorescence generated using a fluorometer or a fluorescent multiwell plate reader. Examples of such fluorescent substances include 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, and coumarin.

Examples of methods for measuring peroxidase activity using the luminescence method include reacting peroxidase with a combination of its substrate, hydrogen peroxide, and a luminescent substance, and measuring the intensity of the luminescence generated using a luminescence spectrometer or luminescence multiwell plate reader. Examples of the luminescent substance include luminol compounds and lucigenin compounds.

When the enzyme is β-D-galactosidase, β-D-galactosidase activity can be measured, for example, by absorbance (colorimetry), luminescence, or fluorescence. An example of a method for measuring β-D-galactosidase activity by absorbance (colorimetry) is to react β-D-galactosidase with its substrate and measure the absorbance of the reaction solution with a spectrophotometer or a multiwell plate reader. An example of a substrate for β-D-galactosidase is o-nitrophenyl-β-D-galactopyranoside. An example of a method for measuring β-D-galactosidase activity by luminescence is to react β-D-galactosidase with its substrate and measure the luminescence intensity of the reaction solution with a luminescence photometer or a luminescence multiwell plate reader. Examples of substrates for β-D-galactosidase include Galacton-Plus (Applied Biosystems) and compounds similar thereto. Methods for measuring β-D-galactosidase activity using a fluorescence method include reacting β-D-galactosidase with its substrate and measuring the fluorescence intensity of the reaction solution using a fluorometer or a fluorescent multiwell plate reader. Examples of substrates for β-D-galactosidase include 4-methylumbelliferyl-β-D-galactopyranoside.

When the enzyme is luciferase, luciferase activity can be measured, for example, by a luminescence method. A method for measuring luciferase activity by a luminescence method includes, for example, reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence photometer or a luminescence multiwell plate reader. Examples of substrates for luciferase include luciferin and coelenterazine.

When the labeling substance is other than a fluorescent substance, a luminescent substance, a radioisotope, or an enzyme, a substance that specifically binds to the labeling substance can be labeled with a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, or the like, and the labeling substance that constitutes the labeled steroid hormone in the immune complex can be bound to the labeling substance, and the fluorescent substance, luminescent substance, radioisotope, or enzyme that labels the substance that specifically binds to the labeling substance can be measured by the above-mentioned method. Examples of substances that specifically bind to the labeling substance include antibodies that specifically bind to the labeling substance, and when the labeling substance is biotin, avidins and the like.

The concentration of the labeled steroid hormone contained in the second reagent is not particularly limited as long as it is a concentration that enables the stabilization method of the measurement kit of the embodiment, and may be, for example, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.4 μg/mL or more, 0.5 μg/mL or more, 0.6 μg/mL or more, 0.7 μg/mL or more, 0.8 μg/mL or more, 0.9 μg/mL or more, or 1 μg/mL or more, and may be 2 μg/mL or less, 3 μg/mL or less, 4 μg/mL or less, 5 μg/mL or less, 6 μg/mL or less, 7 μg/mL or less, 8 μg/mL or less, 9 μg/mL or less, 10 μg/mL or less, 15 μg/mL or less, 20 μg/mL or less, ...2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg/mL or less, 2 μg /mL or less, 25 μg/mL or less, 30 μg/mL or less, 35 μg/mL or less, 40 μg/mL or less, 45 μg/mL or less, 50 μg/mL or less, 60 μg/mL or more, 70 μg/mL or less, 80 μg/mL or less, 90 μg/mL or less, 100 μg/mL or less, 150 μg/mL or less, 200 μg/mL or less, 250 μg/mL or less, 300 μg/mL or less, 350 μg/mL or less, 400 μg/mL or less, 450 μg/mL or less, 500 μg/mL or less, 600 μg/mL or less, 700 μg/mL or less, 800 μg/mL or less, 900 μg/mL or less, or 1 mg/mL or less, with 0.1 to 100 μg/mL being preferred.

The concentration of bovine serum albumin contained in the second reagent is not particularly limited as long as it is a concentration that enables the stabilization method of the measurement kit of the embodiment, and may be, for example, 10 g/L or more, 15 g/L or more, 20 g/L or more, 25 g/L or more, 30 g/L or more, 35 g/L or more, 40 g/L or more, 45 g/L or more, or 50 g/L or more.
In addition, it is desirable to set the concentration of bovine serum albumin so that the viscosity is not too high, taking into consideration the possibility of affecting the amount of sampling of the second reagent by a reagent dispensing member of the measuring device when the measuring device is used. In this case, the concentration of bovine serum albumin may be, for example, 30 g/L or less, 35 g/L or less, 40 g/L or less, 45 g/L or less, 50 g/L or less, 55 g/L or less, 60 g/L or less, 65 g/L or less, 70 g/L or less, 75 g/L or less, or 80 g/L or less.
The above numerical ranges can be freely combined. For example, the concentration of bovine serum albumin in the second reagent may be 10 to 30 g/L, or 20 to 30 g/L.

The aqueous medium contained in the second reagent is not particularly limited as long as it is an aqueous medium that enables the stabilization method of the measurement kit of the embodiment, and examples thereof include the above-mentioned deionized water, distilled water, buffer solution, etc., and buffer solution is preferred. The pH of the buffer solution is not particularly limited as long as it enables the stabilization method of the measurement kit of the embodiment and the measurement using the measurement kit of the embodiment, and may be, for example, pH 4 or more, pH 5 or more, pH 6 or more, or pH 7 or more, and may be pH 8 or less or pH 9 or less, and pH 6 to 8 is preferred, and pH 7 to 8 is more preferred.
The buffering agent and its concentration used to prepare the buffer solution are not particularly limited as long as they are an aqueous medium that enables the stabilization method of the measurement kit of the embodiment, and examples thereof include the buffering agents and their concentrations described above.

The second reagent may contain the preservatives, salts, sugars, surfactants, proteins, interfering substance removers, etc., as necessary.

In the stabilization method of the measurement kit of the embodiment, the storage period of the measurement kit of the embodiment is not particularly limited as long as it is a storage period that enables the stabilization method of the measurement kit of the embodiment, and may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, or 1 year or more, and may be 1.5 years or less, 2 years or less, 2.5 years or less, or 3 years or less, with 4 weeks to 1.5 years being preferred.
In the stabilization method of the measurement kit of the embodiment, the storage temperature of the measurement kit of the embodiment is not particularly limited as long as it is a storage temperature that enables the stabilization method of the measurement kit of the embodiment, and may be, for example, 5°C or higher, 10°C or lower, 15°C or lower, 20°C or lower, 25°C or lower, 30°C or lower, 35°C or lower, 40°C or lower, 45°C or lower, or 50°C or lower, with 5°C to 30°C being preferred.

The measurement kit of the embodiment can be used in a method for measuring steroid hormones in a sample, the method including the following steps:

[1] a step of reacting a specimen with a first reagent containing an anti-steroid hormone antibody in an aqueous medium to generate an immune complex 1 consisting of a steroid hormone in the specimen and the anti-steroid hormone antibody;
[2] a step of adding a second reagent containing a labeled steroid hormone to the reaction solution of the step [1] and reacting to generate an immune complex 2 consisting of the labeled steroid hormone and an anti-steroid hormone antibody;
[3] A step of measuring the labeling substance in the immune complex 2 produced in the step [2].

By further including the following step after the step [3], the concentration of a steroid hormone in a sample can be determined.
[4] carrying out steps [1] to [3] above using a steroid hormone of known concentration instead of a sample, and creating a calibration curve showing the relationship between the steroid hormone concentration and the measured value of the labeled substance; and [5] determining the concentration of the steroid hormone in the sample from the calibration curve created in step [4] and the measured value of the labeled substance measured in step [3].

The specimen, steroid hormone, aqueous medium, anti-steroid hormone antibody, labeled steroid hormone, and labeled substance in the measurement method are not particularly limited as long as they enable measurement using the measurement kit of the embodiment, and examples thereof include the specimen, steroid hormone, aqueous medium, anti-steroid hormone antibody labeled steroid hormone, and labeled substance.

In the measurement method, the concentrations of the aqueous medium, anti-steroid hormone antibody, labeled steroid hormone, and labeled substance in the reaction solution are not particularly limited as long as they are concentrations that enable measurement using the measurement kit of the embodiment, and can be set appropriately according to the concentrations in the first reagent and the second reagent, and the mixing ratio of the sample to the first reagent and the second reagent.

The reaction temperature in the reaction in steps [1] and [2] in the measurement method is not particularly limited as long as it is a temperature that allows measurement using the measurement kit of the embodiment, and is usually 0 to 50°C, preferably 4 to 45°C, and particularly preferably 20 to 40°C. In addition, the reaction time of the reaction is not particularly limited as long as it is a temperature that allows measurement using the measurement kit of the embodiment, and is usually 1 minute to 2 hours, and preferably 2 minutes to 1 hour.

When the measurement kit of the embodiment containing bovine serum albumin is used as is in the measurement method, bovine serum albumin is present in the antibody [1] and/or step [2]. In this case, the bovine serum albumin is not particularly limited as long as it is a bovine serum albumin that allows measurement using the measurement kit of the embodiment, and examples thereof include the bovine serum albumin described above.

(2) Kit for measuring steroid hormones in a sample by a competitive method The kit for measuring steroid hormones in a sample by a competitive method of the embodiment (measurement kit of the embodiment) is a kit for measuring steroid hormones in a sample by a competitive method comprising: a first reagent containing an anti-steroid hormone antibody; and a second reagent containing an aqueous medium, a labeled steroid hormone, and 10 g/L or more of bovine serum albumin, and is, for example, a kit used in the method for measuring steroid hormones in a sample.

The specimen, steroid hormone, first reagent, anti-steroid hormone antibody, second reagent containing bovine serum albumin, aqueous medium, and labeled steroid hormone in the measurement kit of the embodiment are not particularly limited as long as they enable measurement using the measurement kit of the embodiment, and examples thereof include the specimen, steroid hormone, first reagent, anti-steroid hormone antibody, second reagent containing bovine serum albumin, aqueous medium, and labeled steroid hormone.

The concentrations of the anti-steroid hormone antibody, the labeled steroid hormone, the bovine serum albumin, and the buffer used to prepare the buffer solution as the aqueous medium in the measurement kit of the embodiment are not particularly limited as long as they are concentrations that enable measurement using the measurement kit of the embodiment, and examples of such concentrations include the concentrations of the anti-steroid hormone antibody, the labeled steroid hormone, the bovine serum albumin, and the buffer solution used to prepare the buffer solution as the aqueous medium in the measurement kit of the above embodiment.
The pH of the buffer solution as the aqueous medium in the measurement kit of the embodiment is not particularly limited as long as it is a pH that allows measurement using the measurement kit of the embodiment, and examples thereof include the pHs mentioned above.

(3) Method for stabilizing a labeled steroid hormone In one embodiment, the method for stabilizing a labeled steroid hormone is characterized in that the labeled steroid hormone and 10 g/L or more of bovine serum albumin are allowed to coexist in an aqueous medium, and the labeled steroid hormone is used in the method for measuring steroid hormones in a sample.

In the embodiment of the method for stabilizing a labeled steroid hormone, "stable" means that even when the labeled steroid hormone is stored for a long period of time and then used in the method for measuring a steroid hormone in a sample, the signal derived from the labeled substance in the immune complex 2 generated in step [2] of the measurement method is less likely to decrease compared to before storage; for example, even when the labeled steroid hormone is stored at 5°C for 140 days and then used in the measurement method, the signal derived from the labeled substance in the immune complex 2 is 70% or more compared to before storage.
The signal derived from the labeling substance in the immune complex 2 can be measured, for example, by the following method.

[1] A step of reacting a sample not containing steroid hormone with an anti-steroid hormone antibody and an aqueous medium containing a labeled steroid hormone to produce an immune complex 2 consisting of the labeled steroid hormone and the anti-steroid hormone antibody; and [2] a step of measuring the labeled substance in the immune complex 2 produced in step [1].

The signal derived from the labeling substance is measured before and after long-term storage of the labeled steroid hormone of the embodiment, and the ratio of the signal after storage to the signal before storage is calculated. Here, for example, if the ratio is higher than 70%, the measurement kit of the embodiment can be evaluated as being stable.

The labeled steroid hormone and its concentration in the method for stabilizing a labeled steroid hormone of the embodiment are not particularly limited as long as they are capable of stabilizing a labeled steroid hormone of the embodiment, and examples thereof include the labeled steroid hormones and their concentrations described above.

In the method for stabilizing a labeled steroid hormone according to the embodiment, the labeled steroid hormone is made to coexist with bovine serum albumin in an aqueous medium.
The concentration of bovine serum albumin is not particularly limited as long as it is a concentration that enables the stabilization method of the labeled steroid hormone of the embodiment, and examples thereof include the above-mentioned concentrations of bovine serum albumin.

The aqueous medium is not particularly limited as long as it enables the method for stabilizing a labeled steroid hormone of the embodiment, and examples thereof include the above-mentioned deionized water, distilled water, buffer solution, etc., with buffer solution being preferred. The pH of the buffer solution is also not particularly limited as long as it enables the method for stabilizing a labeled steroid hormone of the embodiment and the above-mentioned method for measuring a steroid hormone in a sample, and may be, for example, pH 4 or more, pH 5 or more, pH 6 or more, or pH 7 or more, or may be pH 8 or less, or pH 9 or less, with pH 6 to 8 being preferred, and pH 7 to 8 being more preferred.
The buffering agent and its concentration used to prepare the buffer solution are not particularly limited as long as they are an aqueous medium that enables the stabilization method of the labeled steroid hormone of the embodiment, and examples thereof include the buffering agents and their concentrations described above.

The aqueous medium may contain the above-mentioned anti-steroid hormone antibodies, insoluble carriers, preservatives, salts, sugars, surfactants, proteins, interfering substance removers, etc., as necessary.

In the embodiment of the method for stabilizing a labeled steroid hormone, the storage period of the labeled steroid hormone is not particularly limited as long as it is a storage period that enables the embodiment of the method for stabilizing a labeled steroid hormone, and may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, or 1 year or more, and may be 1.5 years or less, 2 years or less, 2.5 years or less, or 3 years or less, with 4 weeks to 1.5 years being preferred.
In the embodiment of the method for stabilizing a labeled steroid hormone, the storage temperature of the labeled steroid hormone is not particularly limited as long as it is a storage temperature that enables the embodiment of the method for stabilizing a labeled steroid hormone, and may be, for example, 5°C or higher, and 10°C or lower, 15°C or lower, 20°C or lower, 25°C or lower, 30°C or lower, 35°C or lower, 40°C or lower, 45°C or lower, or 50°C or lower, with 5°C to 30°C being preferred.

(4) Labeled steroid hormone-containing reagent In an embodiment, the labeled steroid hormone-containing reagent is a labeled steroid hormone-containing reagent used in a method for measuring a steroid hormone in a sample by a competitive method, in which a steroid hormone in the sample, an anti-steroid hormone antibody, and a labeled steroid hormone are reacted with each other to form an immune complex between the anti-steroid hormone antibody and the labeled steroid hormone, and the label in the immune complex formed is measured, characterized in that the labeled steroid hormone and 10 g/L or more of bovine serum albumin are coexisted in an aqueous medium.

The specimen, steroid hormone, anti-steroid hormone antibody, labeled steroid hormone, and aqueous medium in the labeled steroid hormone-containing reagent of the embodiment are not particularly limited as long as they enable the method of measuring the steroid hormone in the specimen, and examples thereof include the specimen, steroid hormone, anti-steroid hormone antibody, labeled steroid hormone, and aqueous medium.

The concentrations of the anti-steroid hormone antibody, the labeled steroid hormone, the bovine serum albumin, and the buffer used to prepare the buffer solution as the aqueous medium in the labeled steroid hormone-containing reagent of the embodiment are not particularly limited as long as they enable the above-mentioned method for measuring steroid hormones in a sample, and examples of such concentrations include the concentrations of the anti-steroid hormone antibody, the labeled steroid hormone, the bovine serum albumin, and the buffer used to prepare the buffer solution as the aqueous medium in the labeled steroid hormone-containing reagent.
The pH of the buffer solution as the aqueous medium in the labeled steroid hormone-containing reagent of the embodiment is not particularly limited as long as it enables the above-mentioned method for measuring the steroid hormone in the sample, and examples thereof include the above-mentioned pHs.

The aqueous medium may contain the above-mentioned anti-steroid hormone antibodies, insoluble carriers, preservatives, salts, sugars, surfactants, proteins, interfering substance removers, etc., as necessary.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Information on the reagents and equipment used is shown below.
"Biotinylated antibody" solution, "SA-bound particle" solution, "Standard aldosterone reagent A", "Standard aldosterone reagent B", "Standard aldosterone reagent C" and "Standard aldosterone reagent D" from Determiner CL Aldosterone (a kit for measuring aldosterone in a sample by a competitive method; manufactured by Hitachi Chemical Diagnostics Systems), PIPES (manufactured by Dojindo Laboratories), MOPS (manufactured by Dojindo Laboratories), Tween 20 (polyoxyethylene sorbitan monolaurate; manufactured by Tokyo Chemical Industry Co., Ltd.), bovine serum albumin (manufactured by BOVOGEN), and fully automated chemiluminescence immunoassay device CL-JACK NX (manufactured by Hitachi Chemical Diagnostics Systems).

Example 1
The following aldosterone measurement kits A, B, C, D, and E were prepared, each consisting of a first reagent containing a biotin-conjugated anti-aldosterone antibody, a second reagent containing alkaline phosphatase-labeled aldosterone, and a third reagent containing streptavidin-conjugated magnetic particles.

<First reagent: biotin-conjugated anti-aldosterone antibody solution>
A "biotinylated antibody" solution of Determiner CL Aldosterone was prepared as a first reagent containing a biotin-bound anti-aldosterone antibody in a kit for measuring aldosterone in a sample by a competitive method.

<Second Reagent: Alkaline Phosphatase-Labeled Aldosterone Solution>
ALDOSTERONE 3-CMO (aldosterone 3-carboxymethyloxime; STERALOIDS) was mixed with Sulfo-NHS (Thermo Fisher Scientific) and EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; Thermo Fisher Scientific] in a 3:1 mixed solvent of DMSO (Sigma-Aldrich) and water, and reacted at 25° C. for 15 minutes, and then further reacted with alkaline phosphatase (Roche Diagnostics) for 16 hours at 4° C. The reaction mixture was subjected to a Sephadex G-50 column (GE Healthcare Japan) to remove unreacted ALDOSTERONE 3-CMO, and alkaline phosphatase-labeled aldosterone was obtained. Using the obtained alkaline phosphatase-labeled aldosterone, a second reagent having the following composition was prepared.

PIPES (pH see Table 1 or Table 2) 30 mmol/L
Alkaline phosphatase-labeled aldosterone 1.5 μg/mL
Tween20 10g/L
Magnesium chloride hexahydrate 30 mmol/L
Bovine serum albumin (concentration shown in Table 1 or Table 2)

<Third Reagent: Streptavidin-bound Magnetic Particle Suspension>
A solution of "SA-bound particles" of Determiner CL Aldosterone was prepared as a third reagent containing streptavidin-bound magnetic particles in a kit for measuring aldosterone in a sample by a competitive method.

(Comparative Example 1)
A first reagent containing a biotin-conjugated anti-aldosterone antibody, a second reagent containing alkaline phosphatase-labeled aldosterone, and a third reagent containing streptavidin-conjugated magnetic particles were prepared in the same manner as in the second reagent of the aldosterone measurement kit A in Example 1, except that the concentration of bovine serum albumin in the second reagent was 6 g/L, and kit a consisting of the first reagent, the second reagent, and the third reagent was prepared.

Example 2
The stability of the aldosterone measurement kit A of Example 1 was evaluated by the following steps.
(1) Determination of signals derived from labeled substances in the kit immediately after preparation Measurement was performed using a fully automated chemiluminescence immunoassay device CL-JACK NX. A solution (aldosterone concentration: 0 pg/mL, hereinafter referred to as standard A) (10 μL) of standard aldosterone reagent A (lyophilized product) dissolved in 0.5 mL of purified water and a first reagent (50 μL) containing biotin-bound anti-aldosterone antibody of Example 1 immediately after preparation were added to a reaction vessel, stirred, and reacted at 37° C. for 18 minutes. Then, a third reagent (30 μL) containing streptavidin-bound magnetic particles of Example 1 immediately after preparation and a second reagent (30 μL) containing alkaline phosphatase-labeled aldosterone of Kit A of Example 1 immediately after preparation were added to the reaction solution of the reaction, stirred, and reacted at 37° C. for 8 minutes. The magnetic particles were collected by magnetic force, and the reaction solution other than the magnetic particles was removed, and then a washing solution [50 mmol/L MOPS buffer (pH 7.35) containing 0.1% Tween 20] was added to the reaction vessel to wash the magnetic particles. The magnetic particles were collected by magnetic force, and the solution other than the magnetic particles was removed, and then a washing solution was added to the reaction vessel to wash the magnetic particles. After this series of operations of magnetic collection, removal of the solution other than the magnetic particles, and washing the magnetic particles with the washing solution were performed five times, a luminescent substrate solution (70 μL) mainly composed of 9-(4-chlorophenylthiophosphoryloxymethylidene)-10-methylacridan disodium salt was added and stirred, and the amount of luminescence (RLU) generated was measured, and the amount of luminescence when measuring standard A in kit A immediately after preparation was determined.
In addition, the luminescence intensity during measurement of each of the standards B, C, and D in the kit A immediately after preparation was determined by the same method, except that a solution prepared by dissolving standard aldosterone reagent B (lyophilized product) in 0.5 mL of purified water (aldosterone concentration: 50.1 pg/mL, hereinafter referred to as standard B), a solution prepared by dissolving standard aldosterone reagent C (lyophilized product) in 0.5 mL of purified water (aldosterone concentration: 196.6 pg/mL, hereinafter referred to as standard C), and a solution prepared by dissolving standard aldosterone reagent D (lyophilized product) in 0.5 mL of purified water (aldosterone concentration: 1653.9 pg/mL, hereinafter referred to as standard D) were used instead of standard A.

(2) Long-term storage of the kit After the measurement in step (1), the kit A of Example 1 was stored at 5° C. for 4 weeks.
(3) Determination of signals derived from labeled substances in kits after long-term storage The luminescence intensity was measured in the same manner as in step (1) except that the first, second and third reagents stored at 5° C. for 4 weeks in step (2) were used instead of the first, second and third reagents immediately after their preparation, and the luminescence intensity during measurement of each of the standards A, B, C and D in kit A after long-term storage was determined.
(4) Evaluation of kit stability The ratio of the amount of luminescence determined in step (3) when measuring standard A in kit A after long-term storage to the amount of luminescence determined in step (1) when measuring standard A immediately after preparation was taken as 100, i.e., the residual rate of the amount of luminescence when measuring standard A, was calculated. In addition, the residual rates of the amount of luminescence when measuring each of standards B, C, and D were calculated by the same method. The average value of the residual rates of the amount of luminescence obtained when measuring each of standards A, B, C, and D was taken as the residual rate of the signal derived from the labeling substance in kit A, and the stability of kit A was evaluated. The results are shown in Table 1.

Example 3
The stability of Kits B to D was evaluated in the same manner as in Example 2, except that Kits B to D of Example 1 were used instead of Kit A for measuring aldosterone in Example 1. The results are shown in Table 1.

(Comparative Example 2)
The stability of kit a was evaluated in the same manner as in Example 2, except that kit a of the comparative example was used instead of aldosterone measurement kit A of Example 1. The results are shown in Table 1.

As is clear from Table 1, when Kit A of Comparative Example 1 in which the concentration of bovine serum albumin in the second reagent was 6 g/L was used, the remaining rate of the signal derived from the labeling substance was high, being 70% or more, when Kits A to D of Example 1 in which the concentration of bovine serum albumin in the second reagent was 10 g/L or more were used.
Therefore, it has become clear that a kit for measuring steroid hormones in a sample, which comprises a first reagent containing an anti-steroid hormone antibody and the second reagent containing a labeled steroid hormone, is stabilized by causing the labeled steroid hormone and 10 g/L or more of bovine serum albumin to coexist in an aqueous medium in the second reagent.
As is clear from Table 1, the kit D of Example 1, in which the second reagent containing alkaline phosphatase-labeled aldosterone has a pH of 6.5 and the concentration of bovine serum albumin in the second reagent is 6 g/L, shows a high residual rate of the signal derived from the labeling substance, 80% or more, compared with the kit a of Comparative Example 1, in which the second reagent has a pH of 7 or more and the concentration of bovine serum albumin in the second reagent is 10 g/L or more. Therefore, it was revealed that the kit for measuring steroid hormones in a sample, which contains a first reagent containing an anti-steroid hormone antibody and the second reagent containing a labeled steroid hormone, is stabilized by adjusting the pH of the second reagent to 7 or more and allowing the labeled steroid hormone and 10 g/L or more of bovine serum albumin to coexist in an aqueous medium in the second reagent.

Example 4
The stability of the aldosterone measurement kit C or E of Example 1 was evaluated in the same manner as in Example 2, except that the long-term storage in step (2) was storage at 5° C. for 140 days instead of storage at 5° C. for 4 weeks. The results are shown in Table 2.

(Comparative Example 3)
The stability of kit a was evaluated in the same manner as in Example 4, except that kit a of the comparative example was used instead of aldosterone measurement kit C or E of Example 1. The results are shown in Table 2.

As is clear from Table 2, when Kits C and E of Example 1, in which the concentration of bovine serum albumin in the second reagent containing alkaline phosphatase-labeled aldosterone is 20 g/L or more, are used, the residual rate of the signal derived from the labeled substance is high, at 70% or more, compared with the case of using Kit A of Comparative Example 1, in which the concentration of bovine serum albumin in the second reagent containing alkaline phosphatase-labeled aldosterone is 6 g/L. Therefore, it was revealed that the kit for measuring steroid hormones in a sample, which contains a first reagent containing an anti-steroid hormone antibody and the second reagent containing a labeled steroid hormone, is stabilized by allowing the labeled steroid hormone and bovine serum albumin of 20 g/L or more to coexist in an aqueous medium in the second reagent.

Example 5
The ratio of the amount of luminescence obtained in Example 4 when measuring standard A in kit C or E immediately after preparation was calculated based on the amount of luminescence obtained in Comparative Example 3 when measuring standard A in kit a immediately after preparation. Similarly, the ratio of the amount of luminescence obtained in each measurement of standard B, C, and D was also calculated. The results are shown in Table 3.

In general, since steroid hormones are considered to interact with albumin, when using Kits C and E of Example 1, which have a high concentration of bovine serum albumin in the second reagent, there was a concern that the bovine serum albumin bound to the labeled steroid hormone would be removed together with the reaction solution in the step of collecting the magnetic particles by magnetic force and removing the reaction solution other than the magnetic particles in the measurement, and thus the amount of luminescence (signal derived from the labeling substance) would decrease. However, as is clear from Table 3, compared with the case of using Kit a of Comparative Example 1, in which the concentration of bovine serum albumin in the second reagent containing alkaline phosphatase-labeled aldosterone is 6 g/L, even when using Kits C and E of Example 1, in which the concentration of bovine serum albumin in the second reagent is 20 g/L or more, there was no significant change in the amount of luminescence during the measurement of each standard.
Therefore, it has become clear that by allowing the labeled steroid hormone and 20 g/L or more of bovine serum albumin to coexist in an aqueous medium in the second reagent, a kit for measuring steroid hormones in a sample, which comprises a first reagent containing an anti-steroid hormone antibody and the second reagent containing a labeled steroid hormone, can be stabilized without reducing the amount of luminescence (signal derived from the labeled substance) during measurement.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

The present invention provides a method for stabilizing a kit for measuring steroid hormones in a sample by a competitive method; a kit for measuring steroid hormones in a sample by a competitive method that has excellent stability during long-term storage; a method for stabilizing a labeled steroid hormone; and a labeled steroid hormone-containing reagent that has excellent stability during long-term storage.

Claims (29)

A method for stabilizing a kit for measuring a steroid hormone in a sample by a competitive method, the kit comprising a first reagent containing an anti-steroid hormone antibody and a second reagent containing a labeled steroid hormone, the method comprising:
The method for stabilizing the steroid hormone comprises allowing the labeled steroid hormone and bovine serum albumin at a concentration of 10 g/L or more to coexist in an aqueous medium having a pH of 7 to 8 in the second reagent. The stabilization method according to claim 1, wherein the concentration of the bovine serum albumin is 20 to 30 g/L. The stabilization method according to claim 1 or 2 , wherein the labeled steroid hormone is an enzyme-labeled steroid hormone. 4. The method for stabilization according to claim 3 , wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase. the steroid hormone is aldosterone,
the anti-steroid hormone antibody is an anti-aldosterone antibody,
The method for stabilization according to any one of claims 1 to 4 , wherein the labeled steroid hormone is labeled aldosterone . A method for stabilizing a kit for measuring a steroid hormone in a sample by a competitive method, the kit comprising a first reagent containing an anti-steroid hormone antibody and a second reagent which is a solution containing a labeled steroid hormone, the method comprising:
The second reagent is characterized in that the labeled steroid hormone and 10 g/L or more of bovine serum albumin are allowed to coexist in an aqueous medium;
the steroid hormone is a mineralocorticoid such as aldosterone or fludrocortisone acetate, or a sex hormone such as testosterone, dehydroepiandrosterone, estradiol, estriol, estrone, or progesterone;
A stabilization method, wherein the anti-steroid hormone antibody is an antibody against the steroid hormone, and the labeled steroid hormone is a steroid hormone that has been labeled. the steroid hormone is aldosterone,
the anti-steroid hormone antibody is an anti-aldosterone antibody,
The method for stabilization according to claim 6, wherein the labeled steroid hormone is labeled aldosterone. 8. The method for stabilizing bovine serum albumin according to claim 6, wherein the concentration of the bovine serum albumin is 20 to 30 g/L. a first reagent comprising an anti-steroid hormone antibody;
a second reagent comprising an aqueous medium having a pH of 7 to 8 , a labeled steroid hormone, and bovine serum albumin at 10 g/L or more;
A kit for measuring a steroid hormone in a sample by a competitive method, comprising: The kit according to claim 9 , wherein the concentration of the bovine serum albumin is 20 to 30 g/L. 11. The kit according to claim 9 or 10 , wherein the labeled steroid hormone is a steroid hormone labeled with at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase. the anti-steroid hormone antibody is anti-aldosterone;
the anti-steroid hormone antibody is an anti-aldosterone antibody,
The kit according to any one of claims 9 to 11 , wherein the labeled steroid hormone is labeled aldosterone . a first reagent comprising an anti-steroid hormone antibody;
a second reagent comprising an aqueous medium, a labeled steroid hormone, and bovine serum albumin at 10 g/L or more;
A kit for measuring a steroid hormone in a sample by a competitive method, comprising:
the steroid hormone is a mineralocorticoid such as aldosterone or fludrocortisone acetate, or a sex hormone such as testosterone, dehydroepiandrosterone, estradiol, estriol, estrone, or progesterone;
A kit, wherein the anti-steroid hormone antibody is an antibody against the steroid hormone, and the labeled steroid hormone is a steroid hormone that has been labeled. the steroid hormone is aldosterone,
the anti-steroid hormone antibody is an anti-aldosterone antibody,
The kit of claim 13, wherein the labeled steroid hormone is labeled aldosterone. The kit according to claim 13 or 14, wherein the concentration of the bovine serum albumin is 20 to 30 g/L. A method for stabilizing a labeled steroid hormone, which comprises allowing the labeled steroid hormone to coexist with 10 g/L or more of bovine serum albumin in an aqueous medium having a pH of 7 to 8 . The method for stabilizing an albumin composition according to claim 16 , wherein the concentration of the bovine serum albumin is 20 to 30 g/L. The stabilization method according to claim 16 or 17 , wherein the labeled steroid hormone is a steroid hormone labeled with at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase. the steroid hormone is aldosterone,
The method for stabilization according to any one of claims 16 to 18 , wherein the labeled steroid hormone is labeled aldosterone . 1. A method for stabilizing a labeled steroid hormone in a solution, comprising the steps of: allowing the labeled steroid hormone to coexist with 10 g/L or more of bovine serum albumin in an aqueous medium;
A stabilization method, wherein the labeled steroid hormone is labeled aldosterone, labeled fludrocortisone acetate, labeled testosterone, labeled dehydroepiandrosterone, labeled estradiol, labeled estriol, labeled estrone, or labeled progesterone. The stabilization method according to claim 20, wherein the labeled steroid hormone is labeled aldosterone. The method for stabilization according to claim 20 or 21, wherein the concentration of the bovine serum albumin is 20 to 30 g/L. a method for measuring a steroid hormone in a sample by a competitive method, which comprises reacting a steroid hormone in the sample with an anti-steroid hormone antibody and a labeled steroid hormone to form an immune complex between the anti-steroid hormone antibody and the labeled steroid hormone, and measuring the label in the immune complex thus formed;
A labeled steroid hormone-containing reagent for use in
A labeled steroid hormone-containing reagent, characterized in that a labeled steroid hormone and 10 g/L or more of bovine serum albumin coexist in an aqueous medium having a pH of 7-8 . The reagent according to claim 23 , wherein the concentration of the bovine serum albumin is 20 to 30 g/L. 25. The reagent according to claim 23 or 24 , wherein the labeled steroid hormone is a steroid hormone labeled with at least one enzyme selected from the group consisting of alkaline phosphatase and peroxidase. the steroid hormone is aldosterone,
the anti-steroid hormone antibody is an anti-aldosterone antibody,
The reagent according to any one of claims 23 to 25 , wherein the labeled steroid hormone is labeled aldosterone . a method for measuring a steroid hormone in a sample by a competitive method, which comprises reacting a steroid hormone in the sample with an anti-steroid hormone antibody and a labeled steroid hormone to form an immune complex between the anti-steroid hormone antibody and the labeled steroid hormone, and measuring the label in the immune complex thus formed;
A labeled steroid hormone-containing reagent for use in
The method is characterized in that a labeled steroid hormone and bovine serum albumin of 10 g/L or more are present together in an aqueous medium,
the steroid hormone is a mineralocorticoid such as aldosterone or fludrocortisone acetate, or a sex hormone such as testosterone, dehydroepiandrosterone, estradiol, estriol, estrone, or progesterone;
The reagent , wherein the anti-steroid hormone antibody is an antibody against the steroid hormone, and the labeled steroid hormone is a steroid hormone that has been labeled . the steroid hormone is aldosterone,
the anti-steroid hormone antibody is an anti-aldosterone antibody,
28. The reagent of claim 27, wherein the labeled steroid hormone is labeled aldosterone. The reagent according to claim 27 or 28, wherein the concentration of the bovine serum albumin is 20 to 30 g/L.