CN112394182A

CN112394182A - Estradiol release reagent and detection kit thereof

Info

Publication number: CN112394182A
Application number: CN202011187171.1A
Authority: CN
Inventors: 谢钧; 于琴; 丁军发; 汪紫晶
Original assignee: Shenzhen Chenna Biotechnology Co ltd
Current assignee: Shenzhen Chenna Biotechnology Co ltd
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-02-23

Abstract

An estradiol releasing reagent and a detection kit thereof, the estradiol releasing reagent being capable of affecting the binding of estradiol to sex hormone binding protein to release estradiol, the estradiol releasing reagent containing at least one of a reagent for competitively binding to sex hormone binding protein and a reagent for reducing the binding force of estradiol to sex hormone binding protein. Compared with the prior art, the estradiol releasing agent is used as a component of an estradiol enzymatic chemiluminescence immunoassay reagent, and can rapidly and effectively dissociate estradiol from albumin and sex hormone binding protein.

Description

Estradiol release reagent and detection kit thereof

Technical Field

The invention relates to the technical field of steroid estrogen release, in particular to an estradiol release reagent and a detection kit thereof.

Background

Estradiol is a steroid estrogen, derived from cholesterol in humans and higher animals. Cholesterol is oxidized to generate pregnenolone, and then progesterone and 17 alpha-hydroxyprogesterone are generated from the pregnenolone until the intermediate androstenedione. Androstenedione is partly converted into testosterone and into estradiol by aromatase, and partly into estrone by aromatization and subsequently into estradiol. Estradiol, a major female hormone, is responsible for regulation of female characteristics, the maturation of accessory sexual organs, and the menstrual-ovulation cycle. Estradiol may be produced by granulosa cells, placenta, theca cells, leydig cells and adrenal cortex cells. For women of childbearing age, estradiol is produced primarily by the metabolism of androstenedione produced by follicular cells of the follicular membrane by the ovarian granulosa cells. The level of serum estradiol reflects the activity of female ovary, and is helpful for diagnosing amenorrhea, menstrual dysfunction, estrogen-producing tumor, precocious puberty and other diseases.

In plasma, estradiol is bound predominantly to sex hormone binding proteins and albumin, with a small fraction of free bioactive estradiol remaining in constant proportion throughout the menstrual cycle. The immunoassay for estradiol requires that estradiol be released from the sex hormone binding protein, albumin, in order to detect antibody recognition. Therefore, the formula composition of the estradiol releasing agent is related to the releasing effect of the estradiol, and the sensitivity and the specificity of the estradiol immunoassay reagent are directly influenced. The existing estradiol releasing agent is difficult to sufficiently dissociate estradiol from albumin and sex hormone binding protein.

Disclosure of Invention

The invention aims to provide an estradiol release reagent and a detection kit thereof.

According to a first aspect, there is provided in one embodiment an estradiol releasing agent capable of affecting the binding of estradiol to sex hormone binding protein so as to release estradiol, the estradiol releasing agent comprising at least one of an agent for competitively binding to sex hormone binding protein, an agent for reducing the binding of estradiol to sex hormone binding protein;

the agent for competitively binding sex hormone binding protein comprises at least one of dihydrotestosterone, danazol, mesterone, testosterone, cortisol, 3, 4-Divanilyltetrahydrofuran (DVT), levonorgestrel;

the agent for reducing the binding force of estradiol to sex hormone binding protein comprises at least one of heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, and sodium trichloroacetate.

According to a second aspect, in one embodiment there is provided a test kit comprising an estradiol releasing reagent according to the first aspect.

According to a third aspect, there is provided in one embodiment a method of detecting estradiol using the test kit of the second aspect, comprising: mixing a sample to be detected, the estradiol release reagent, the estradiol magnetic separation reagent, the estradiol antibody reagent and the estradiol antigen reagent for immunoreaction, wherein the obtained mixed solution contains at least one of magnetic microspheres with the concentration of 0.0025-0.0125 wt%, estradiol antibodies with the concentration of 0.5-2.5 mu g/mL, enzyme-labeled estradiol antigens with the concentration of 0.125-1.0 mu g/mL and the estradiol release agent with the following concentration:

0.025-0.125mg/mL dihydrotestosterone, 0.0125-0.075mg/mL danazol, 0.005-0.0625mg/mL mesterone, 0.0025-0.05mg/mL testosterone, 2.5-4.0mg/mL heparin sodium, 0.005-0.025mg/mL cortisol, 1.25-5mg/mL 8-aniline-1-naphthalene sulfonic acid, 0.25-5mg/mL sodium trichloroacetate, 0.0125-0.0625mg/mL 3, 4-divanilyltetrahydrofuran, 0.05-0.1mg/mL levonorgestrel.

According to a fourth aspect, there is provided in one embodiment the use of an estradiol release reagent according to the first aspect or a test kit according to the second aspect for the detection of estradiol.

According to the estradiol releasing agent of the embodiment, compared with the prior art, the estradiol releasing agent as a component of the estradiol enzymatic chemiluminescence immunoassay reagent can rapidly and effectively dissociate estradiol from albumin and sex hormone binding protein, and the estradiol detection efficiency is improved.

Drawings

FIG. 1 is a graph showing the results of estradiol release in a sample without release agent;

FIG. 2 is a graph showing the results of estradiol release in samples treated with the release agent 1;

FIG. 3 is a graph showing the results of estradiol release in samples treated with the release agent 2;

FIG. 4 is a graph showing the results of estradiol release in samples treated with the release agent 3;

fig. 5 is a graph showing the results of estradiol release in samples treated with the release agent 4.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

Herein, unless otherwise indicated, estradiol is also referred to as E2.

In a first aspect, in one embodiment, an estradiol releasing reagent is provided that may be used to make up an estradiol enzymatic chemiluminescent immunoassay kit.

In one embodiment, an estradiol releasing agent is provided that includes a compound capable of promoting the release of estradiol from sex hormone binding protein, albumin, and cortisol binding globulin.

In one embodiment, an estradiol releasing agent is provided that is capable of affecting the binding of estradiol to sex hormone binding protein, thereby releasing estradiol, the estradiol releasing agent comprising at least one of an agent for competitive binding to sex hormone binding protein, an agent for reducing the binding of estradiol to sex hormone binding protein, the agent for competitive binding to sex hormone binding protein comprising at least one of dihydrotestosterone, danazol, mesterone, testosterone, cortisol, 3, 4-Divanilyltetrahydrofuran (DVT), levonorgestrel; the agent for reducing the binding force of estradiol to sex hormone binding protein comprises at least one of heparin sodium, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate, and sodium trichloroacetate.

Dihydrotestosterone, danazol, mesterone, testosterone, cortisol, 3, 4-Divanilyltetrahydrofuran (DVT), levonorgestrel act to competitively bind to sex hormone binding proteins, allowing estradiol to be released. The sodium heparin, 8-aniline-1-naphthalene sulfonic Acid (ANS), 8-aniline-1-naphthalene sulfonate and sodium trichloroacetate can reduce the binding force of estradiol and sex hormone binding protein, so that estradiol is released.

In one embodiment, testosterone is also known as testosterone.

In one embodiment, 3, 4-divanilyltetrahydrofuran has a CAS number of 34730-78-4 and a formula of C₂₀H₂₄O₅Abbreviated DVT.

In one embodiment, the combination of an agent that competitively binds to sex hormone binding protein and an agent that reduces estradiol binding to sex hormone binding protein act synergistically to allow for adequate release of estradiol and for a significant increase in the sensitivity and specificity of the estradiol immunoassay reagent.

In one embodiment, 3, 4-Divanilyltetrahydrofuran (DVT) as a non-hormonal ligand competes for binding to sex hormone binding proteins to release estradiol, thereby increasing the efficiency of estradiol release.

In one embodiment, the estradiol releasing agent of the present invention is used as a component of the immunological reagent, and the immunological reaction is performed while the estradiol is released, that is, the dissociation process and the immunological reaction process occur simultaneously without a separate dissociation (sample treatment) process.

In one embodiment, an estradiol releasing agent is provided that contains at least one of the following components in the following concentrations: 0.1-0.5mg/mL dihydrotestosterone, 0.05-0.3mg/mL danazol, 0.02-0.25mg/mL mesterone, 0.01-0.2mg/mL testosterone, 10-50mg/mL heparin sodium, 0.02-0.1mg/mL cortisol, 5-20mg/mL 8-aniline-1-naphthalenesulfonic Acid (ANS), 1-20mg/mL sodium trichloroacetate, 0.05-0.25mg/mL 3, 4-Divanilyltetrahydrofuran (DVT), 0.2-0.4mg/mL levonorgestrel. Each concentration herein refers to the concentration of each component throughout the estradiol releasing agent.

In one embodiment, the estradiol releasing agent contains the following components in concentrations: 0.05-0.3mg/mL danazol and 1-20mg/mL sodium trichloroacetate.

In one embodiment, the estradiol releasing agent contains the following components in concentrations: 0.1-0.5mg/mL dihydrotestosterone and 1-20mg/mL sodium trichloroacetate.

In one embodiment, the estradiol releasing agent contains the following components in concentrations: 0.02-0.1mg/mL of cortisol, 0.02-0.25mg/mL of mesterone and 5-20mg/mL of 8-aniline-1-naphthalenesulfonic acid.

In one embodiment, the estradiol releasing agent contains the following components in concentrations: 0.05-0.3mg/mL danazol, 0.05-0.25mg/mL 3, 4-divanilyltetrahydrofuran, 5-20mg/mL 8-aniline-1-naphthalenesulfonic acid.

In one embodiment, a buffer is also included.

In one embodiment, the buffer is exemplified by, but not limited to, at least one selected from the group consisting of phosphate buffer, 2-morpholinoethanesulfonic acid buffer, Tris-HCl buffer, citrate buffer, 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO) buffer, glycine-hydrochloric acid buffer, acetic acid-sodium acetate buffer, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and the like. The above buffers are only partially exemplified, and the buffer suitable for use in the present invention is not limited, and may be other buffers having similar functions.

In one embodiment, 2-morpholinoethanesulfonic Acid buffer, abbreviated as MES, is abbreviated as 4-morpholinone Sulfonic Acid, also referred to as 2- (N-morpholino) ethanesulfonic Acid, and is a buffer having a buffering pH range of 5.5-6.7, which is close to physiological pH.

In one embodiment, a solvent is also included.

In one embodiment, the solvent may be selected from at least one of an organic solvent and an inorganic solvent.

In one embodiment, the solvent may be selected from at least one of dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), water.

In one embodiment, the water may be pure water, i.e., deionized water.

In one embodiment, a method of preparing an estradiol releasing agent comprises: taking the original components of the estradiol releasing agent with required amount, dissolving the original components respectively by using solvents such as DMSO, DMF or pure water and the like, preparing mother liquor of 500mg/mL, and diluting the mother liquor to corresponding concentration by using buffer solution to prepare the estradiol releasing agent.

In one embodiment, the delivery agent is used as a component of an estradiol enzymatic chemiluminescence immunoassay reagent, and can rapidly and effectively dissociate estradiol from albumin and sex hormone binding protein, so that the sensitivity and specificity of the estradiol immunoassay reagent are obviously improved.

In a second aspect, in one embodiment, there is provided a test kit comprising the estradiol releasing reagent of the first aspect.

In one embodiment, the detection kit is an estradiol nanomagnetic particle enzymatic chemiluminescence immunoassay kit.

In one embodiment, the assay kit further comprises magnetic microparticles, an estradiol antibody, an estradiol antigen, an enzymatic chemiluminescent substrate. The estradiol releasing agent of the present invention is suitable for use in a detection reagent without limitation, and may be a magnetic particle chemiluminescence immunoassay reagent of estradiol with other compositions.

In one embodiment, the test kit further comprises a magnetic estradiol segregation reagent (i.e., a reagent comprising magnetic microparticles), an estradiol antibody reagent (i.e., a reagent comprising an estradiol antibody), an estradiol antigen reagent (i.e., a reagent comprising an estradiol antigen), and an enzymatic chemiluminescent substrate reagent (i.e., a reagent comprising an enzymatic chemiluminescent substrate).

In one embodiment, the magnetic particles in the estradiol magnetic separation reagent are selected from at least one of streptavidin superparamagnetic nanoparticles, carboxylated magnetic nanoparticles and tosyl magnetic nanoparticles; the magnetic nanoparticles described above are all commercially available.

In one embodiment, the estradiol antibody in the estradiol antibody agent, the estradiol antigen in the estradiol antigen agent is selected from any one of the following combinations:

1) biotin-labeled estradiol antibodies, enzyme-labeled estradiol antigens;

2) biotin-labeled estradiol antigen, enzyme-labeled estradiol antibody.

In one embodiment, the estradiol release reagent, the estradiol magnetic separation reagent, the estradiol antibody reagent, the estradiol antigen reagent and the enzymatic chemiluminescent substrate are mixed with the sample according to required amounts for immunoreaction before use, and the method comprises the steps of mixing a sample to be detected with the estradiol release reagent, the estradiol magnetic separation reagent, the reagent containing the estradiol antibody and the reagent containing the estradiol antigen for immunoreaction, and adding the enzymatic chemiluminescent substrate reagent for luminous reaction after the reaction is finished.

In one embodiment, the estradiol antibody and estradiol antigen are commercially available.

In one embodiment, the enzyme is selected from at least one of alkaline phosphatase, horseradish peroxidase. The enzyme may be commercially available.

In one embodiment, the enzymatic chemiluminescent substrate includes, but is not limited to, at least one of 3-aminophthalic hydrazide (i.e., luminol), a 1, 2-dioxane derivative, disodium (i.e., APS-5) phosphate salt of (4-chlorobenzenethiol) (10-methyl-9, 10-acridan methylene).

In one embodiment, the 1, 2-dioxetane derivative includes, but is not limited to, any one of AMPPD, CSPD, CDP-STAR, ADP-STAR. Enzymatic chemiluminescent substrates are also commercially available.

In one embodiment, the chinese name of AMPPD: 3- (2-Spiraadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt, CAS: 122341-56-4.

In one embodiment, the concentration of magnetic microspheres in the estradiol magnetic separation reagent is 0.01-0.05 wt%, including but not limited to 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, and the like. This concentration is the initial concentration, or referred to as the dispensed concentration or the stored concentration. Typically, the concentration of each component will change upon mixing the estradiol magnetic separation agent with the estradiol antibody, estradiol antigen, enzymatic chemiluminescent substrate, estradiol releasing agent, i.e., the working concentration of each component will typically be different from the storage concentration.

In one embodiment, the concentration of estradiol antibody in the estradiol antibody reagent is 2-10 μ g/mL, including but not limited to 2 μ g/mL, 3 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL, 10 μ g/mL, and the like. The concentration is the storage concentration.

In one embodiment, a method of making a biotin-labeled estradiol antibody comprises: taking a proper amount of estradiol antibody, diluting the estradiol antibody with phosphate buffer solution with pH8.0 and 0.2mol/L to the final concentration of 5mg/mL, adding biotin activated ester to ensure that the final concentration of the biotin activated ester in the system is 10mg/mL, uniformly mixing, and reacting at room temperature for 3 hours. And purifying the mixed solution in an ultrafiltration and centrifugation mode to obtain the biotin-labeled estradiol antibody. This is merely an exemplary list, and biotin labeling can be carried out using a labeling system other than the above-described labeling system. The biotin-labeled estradiol antibody was diluted with 50mM PBS buffer, pH7.4, to obtain the estradiol antibody reagent at a concentration of 2-10. mu.g/mL.

In one embodiment, the concentration of estradiol antigen in the estradiol antigenic agent is from 0.5 to 4.0 μ g/mL, which is the storage concentration. Specifically, but not limited to, 0.5. mu.g/mL, 0.6. mu.g/mL, 0.7. mu.g/mL, 0.8. mu.g/mL, 0.9. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL, 3.0. mu.g/mL, 4.0. mu.g/mL, and the like can be included.

In one embodiment, the alkaline phosphatase-labeled estradiol antigen is prepared by a method comprising: dissolving appropriate amount of alkaline phosphatase in pure water, adding prepared NaIO₄After the solution is reacted at room temperature in the dark, the reaction solution is added into an ultrafiltration centrifugal tube, and is subjected to ultrafiltration centrifugation at low temperature (such as 4 ℃) and then buffer solution replacement is carried out by using carbonate buffer solution. Adding a certain amount of estradiol-BSA coupling compound into the alkaline phosphatase after buffer solution replacement, adding the prepared sodium borohydride solution after the reaction at room temperature in a dark place, uniformly mixing, and standing at low temperature (such as 4 ℃) for a certain time (such as 2 hours). And after the reaction is finished, performing ultrafiltration centrifugation by using an ultrafiltration centrifugal tube, and performing buffer solution replacement by using a PBS buffer solution to prepare the estradiol antigen marked by the alkaline phosphatase.

In one embodiment, the concentration of enzymatic chemiluminescent substrate in the reagent comprising enzymatic chemiluminescent substrate is 0.02 to 4mmol/L, including but not limited to 0.02mmol/L, 0.04mmol/L, 0.05mmol/L, 0.06mmol/L, 0.08mmol/L, 0.1mmol/L, 0.5mmol/L, 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, 3mmol/L, 3.5mmol/L, 4mmol/L, and the like, at storage concentration.

In a third aspect, in one embodiment, there is provided a method for detecting estradiol using the detection kit of the second aspect, comprising: mixing a sample to be detected, the estradiol release reagent, the estradiol magnetic separation reagent, the estradiol antibody reagent and the estradiol antigen reagent for immunoreaction, wherein the obtained mixed solution contains at least one of magnetic microspheres with the concentration of 0.0025-0.0125 wt%, estradiol antibodies with the concentration of 0.5-2.5 mu g/mL, enzyme-labeled estradiol antigens with the concentration of 0.125-1.0 mu g/mL and the estradiol release agent with the following concentration:

In one embodiment, after completion of the immunological reaction, the enzymatic chemiluminescent substrate reagent is added to provide a reagent useful for estradiol detection (i.e., estradiol detection reagent) having an enzymatic chemiluminescent substrate concentration of 0.01-2.0 g/L.

In one embodiment, the concentration of dihydrotestosterone in the resulting mixture can include, but is not limited to, 0.025mg/mL, 0.03mg/mL, 0.05mg/mL, 0.06mg/mL, 0.075mg/mL, 0.1mg/mL, 0.125mg/mL, and the like.

In one embodiment, the concentration of danazol in the resulting mixture can include, but is not limited to, 0.0125mg/mL, 0.02mg/mL, 0.025mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.075mg/mL, and the like.

In one embodiment, the concentration of mesterone in the resulting mixture can include, but is not limited to, 0.005mg/mL, 0.01mg/mL, 0.015mg/mL, 0.02mg/mL, 0.025mg/mL, 0.03mg/mL, 0.035mg/mL, 0.04mg/mL, 0.045mg/mL, 0.05mg/mL, 0.055mg/mL, 0.06mg/mL, 0.0625mg/mL, and the like.

In one embodiment, the concentration of testosterone in the resulting mixture can include, but is not limited to, 0.0025mg/mL, 0.005mg/mL, 0.0075mg/mL, 0.01mg/mL, 0.0125mg/mL, 0.015mg/mL, 0.0175mg/mL, 0.02mg/mL, 0.0225mg/mL, 0.025mg/mL, 0.0275mg/mL, 0.03mg/mL, 0.0325mg/mL, 0.04mg/mL, 0.0425mg/mL, 0.045mg/mL, 0.0475mg/mL, 0.05mg/mL, and the like.

In one embodiment, the concentration of heparin sodium in the resulting mixture may include, but is not limited to, 2.5mg/mL, 2.6mg/mL, 2.7mg/mL, 2.8mg/mL, 2.9mg/mL, 3mg/mL, 3.1mg/mL, 3.2mg/mL, 3.3mg/mL, 3.4mg/mL, 3.5mg/mL, 3.6mg/mL, 3.7mg/mL, 3.8mg/mL, 3.9mg/mL, 4.0mg/mL, and the like.

In one embodiment, the concentration of cortisol in the resulting mixture may include, but is not limited to, 0.005mg/mL, 0.01mg/mL, 0.015mg/mL, 0.02mg/mL, 0.025mg/mL, and the like.

In one embodiment, the concentration of 8-aniline-1-naphthalenesulfonic acid in the resulting mixture may include, but is not limited to, 1.25mg/mL, 1.3mg/mL, 1.35mg/mL, 1.4mg/mL, 1.45mg/mL, 1.5mg/mL, 1.55mg/mL, 1.6mg/mL, 1.65mg/mL, 1.7mg/mL, 1.75mg/mL, 1.8mg/mL, 1.85mg/mL, 1.9mg/mL, 1.95mg/mL, 2mg/mL, 2.05mg/mL, 2.1mg/mL, 2.15mg/mL, 2.2mg/mL, 2.25mg/mL, 2.3mg/mL, 2.35mg/mL, 2.4mg/mL, 2.5mg/mL, 2.6mg/mL, 2.7mg/mL, 2.8mg/mL, 2.9mg/mL, 3.0mg/mL, 3mg/mL, 3.5mg/mL, 3mg/mL, 1.5mg/mL, 1mg/mL, 1.65mg/mL, 3.2mg/mL, 3.3mg/mL, 3.4mg/mL, 3.5mg/mL, 3.6mg/mL, 3.7mg/mL, 3.8mg/mL, 3.9mg/mL, 4.0mg/mL, 4.1mg/mL, 4.2mg/mL, 4.3mg/mL, 4.4mg/mL, 4.5mg/mL, 4.6mg/mL, 4.7mg/mL, 4.8mg/mL, 4.9mg/mL, 5.0mg/mL, and the like.

In one embodiment, the concentration of sodium trichloroacetate in the resulting mixture may include, but is not limited to, 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1mg/mL, 1.25mg/mL, 1.5mg/mL, 1.75mg/mL, 2mg/mL, 2.25mg/mL, 2.5mg/mL, 2.75mg/mL, 3mg/mL, 3.25mg/mL, 3.5mg/mL, 3.75mg/mL, 4mg/mL, 4.25mg/mL, 4.5mg/mL, 4.75mg/mL, 5mg/mL, and the like.

In one embodiment, the concentration of 3, 4-divanilyltetrahydrofuran in the resulting mixture may include, but is not limited to, 0.0125mg/mL, 0.0250mg/mL, 0.0375mg/mL, 0.0500mg/mL, 0.0625mg/mL, and the like.

In one embodiment, the concentration of levonorgestrel in the resulting mixture may include, but is not limited to, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.1mg/mL, and the like.

In one embodiment, the concentration of the magnetic microspheres in the resulting mixture may include, but is not limited to, 0.0025 wt%, 0.0050 wt%, 0.0075 wt%, 0.01 wt%, 0.0125 wt%, and the like.

In one embodiment, the concentration of estradiol antibody in the resulting mixture may include, but is not limited to, 0.5. mu.g/mL, 0.6. mu.g/mL, 0.7. mu.g/mL, 0.8. mu.g/mL, 0.9. mu.g/mL, 1. mu.g/mL, 1.1. mu.g/mL, 1.2. mu.g/mL, 1.3. mu.g/mL, 1.4. mu.g/mL, 1.5. mu.g/mL, 1.6. mu.g/mL, 1.7. mu.g/mL, 1.8. mu.g/mL, 1.9. mu.g/mL, 2. mu.g/mL, 2.1. mu.g/mL, 2.2. mu.g/mL, 2.3. mu.g/mL, 2.4. mu.g/mL, 2.5. mu.g/mL, and the like.

In one embodiment, the concentration of estradiol antigen in the resulting mixture may include, but is not limited to, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.375. mu.g/mL, 0.5. mu.g/mL, 0.625. mu.g/mL, 0.75. mu.g/mL, 0.875. mu.g/mL, 1. mu.g/mL, and the like.

In one embodiment, after addition of the enzymatic chemiluminescent substrate reagent, the concentration of enzymatic chemiluminescent substrate in the resulting reagent may include, but is not limited to, 0.01g/L, 0.02g/L, 0.03g/L, 0.04g/L, 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1.0g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L, 2g/L, etc.

The concentrations of the components described above are merely exemplary and other concentrations of the components are possible.

In a fourth aspect, in one embodiment, there is provided the use of an estradiol release reagent according to the first aspect or a test kit according to the second aspect for the detection of estradiol.

The pH in the following examples was adjusted with an aqueous acid or alkali solution, the acid being hydrochloric acid and the alkali being an aqueous sodium hydroxide solution.

Examples 1 to 4

1. Sample releasing agent 1: contains 100. mu.g/mL danazol, 2mg/mL sodium trichloroacetate, 50mM MES buffer, pH 6.0.

2. Sample releasing agent 2: contains 200 mu g/mL dihydrotestosterone, 2mg/mL sodium trichloroacetate and 100mM MOPSO buffer solution, and has a pH value of 6.5.

3. Sample releasing agent 3: contains 0.05mg/mL cortisol, 0.1mg/mL mesterone, 6mg/mL 8-aniline-1-naphthalenesulfonic Acid (ANS), and 60mM Tris-HCl buffer, and the pH is adjusted to 8.5.

4. Sample releasing agent 4: contains 50. mu.g/mL danazol, 200. mu.g/mL 3, 4-Divanilyltetrahydrofuran (DVT), 5mg/mL 8-aniline-1-naphthalenesulfonic Acid (ANS), 50mM PBS buffer, pH 7.4.

In this example, the preparation method of the estradiol magnetic separation reagent is as follows: and (3) taking commercially available streptavidin magnetic particles, and diluting the streptavidin magnetic particles to a final concentration of 0.01 wt% by using 0.05mol/L Tris-HCl buffer solution to obtain the magnetic separation reagent of estradiol.

In this example, the biotin-labeled estradiol antibody was prepared as follows: taking a proper amount of estradiol antibody, diluting the estradiol antibody with phosphate buffer solution with pH8.0 and 0.2mol/L until the final concentration is 5mg/mL, adding biotin activated ester to ensure that the final concentration of the biotin activated ester in a system is 10mg/mL, uniformly mixing, and reacting at room temperature for 3 hours. And purifying the mixed solution in an ultrafiltration and centrifugation mode to obtain the biotin-labeled estradiol antibody. The biotin-labeled estradiol antibody was diluted with 50mM PBS buffer, pH7.4, to obtain the estradiol antibody reagent at a concentration of 2.5. mu.g/mL.

In this example, the preparation method of the alkaline phosphatase-labeled estradiol antigen reagent was as follows: dissolving appropriate amount of alkaline phosphatase in 0.8mL of purified water, adding 0.2mL of prepared 0.1M NaIO to alkaline phosphatase₄The solution is reacted for 20min at room temperature in a dark place, the buffer solution is added into a 10KD ultrafiltration centrifugal tube, ultrafiltration and centrifugation are carried out at 4 ℃, and the buffer solution is replaced by carbonate buffer solution with pH of 9.5 and 0.2M. To the alkaline phosphatase after the buffer replacement, a certain amount of estradiol-BSA conjugate was added, and the reaction was carried out at room temperature for 3 hours in the absence of light. 0.1mL of the prepared 4mg/mL sodium borohydride solution is added, and the mixture is allowed to stand for 2 hours at 4 ℃ after being uniformly quenched. After the reaction, the mixture was centrifuged by ultrafiltration using a 10kD centrifugal tube and then subjected to buffer exchange with 50mM PBS buffer (pH7.4) to prepare an alkaline phosphatase-labeled estradiol antigen, and then the alkaline phosphatase-labeled estradiol antigen was added to the mixture with a composition of 50mM PBS and 4mM MgCl₂、2mM ZnCl₂0.05% PC300 (bacteriostatic agent) and a reagent having a pH of 7.0, to obtain an estradiol antigen reagent of 1.0. mu.g/mL.

In this example, enzymatic chemiluminescent substrates for alkaline phosphatase were prepared as follows: 50mM Tris, 0.5mM APS-5, 5mM MgCl were prepared in ultrapure water₂、2.5mM ZnCl₂、0.1％TritonX100、0.05％PrA solution of oclin 300 (preservative) was adjusted to pH 9.5 with hydrochloric acid to prepare a reagent containing an enzymatic chemiluminescent substrate.

APS-5 alkaline phosphatase chemiluminescent substrates were purchased from Wuxi Baimeige Biotech, Inc., cat #: BMALP-100.

In this example, the rogowski estradiol assay kit (electrochemiluminescence method) was purchased from rogowski diagnostic products (shanghai) ltd, and the manufacturer was germany rogowski diagnostic ltd, product name: estradiol detection kit (electrochemiluminescence).

Specificity: a total of 60 clinical serum samples of male and female are respectively taken, and assigned and determined by a Roche estradiol detection kit (electrochemical luminescence method). The samples were then tested separately with immunoassay kits containing the 4 different sample release agents described above.

The immunoassay kit without release agent (single part) consists of the following components: 100. mu.L of a buffer (i.e., a control buffer, specifically 50mM PBS buffer, pH 7.4) for preparing a releasing agent, 60. mu.L of a estradiol magnetic separation reagent, 100. mu.L of a biotin-labeled estradiol antibody reagent, 80. mu.L of an alkaline phosphatase-labeled estradiol antigen reagent, and 200. mu.L of a reagent containing an enzymatic chemiluminescent substrate.

The immunoassay kit (single person) containing the sample releasing agent 1 consists of: 1100 mu L of sample releasing agent, 60 mu L of estradiol magnetic separation reagent, 100 mu L of biotin-labeled estradiol antibody reagent, 80 mu L of alkaline phosphatase-labeled estradiol antigen reagent and 200 mu L of reagent containing enzymatic chemiluminescence substrate.

The immunoassay kit (single person) containing the sample releasing agent 2 consists of: 2100 ul of sample release agent, 60 ul of estradiol magnetic separation reagent, 100 ul of biotin-labeled estradiol antibody reagent, 80 ul of alkaline phosphatase-labeled estradiol antigen reagent, and 200 ul of reagent containing enzymatic chemiluminescent substrate.

The immunoassay kit (single person) containing the sample releasing agent 3 consists of: 3100 μ L of sample releasing agent, 60 μ L of estradiol magnetic separation agent, 100 μ L of biotin-labeled estradiol antibody agent, 80 μ L of alkaline phosphatase-labeled estradiol antigen agent, and 200 μ L of enzyme-catalyzed chemiluminescent substrate-containing agent.

The immunoassay kit (single person) containing the sample releasing agent 4 consists of: 4100. mu.L of sample release agent, 60. mu.L of estradiol magnetic separation agent, 100. mu.L of biotin-labeled estradiol antibody agent, 80. mu.L of alkaline phosphatase-labeled estradiol antigen agent, and 200. mu.L of enzyme-catalyzed chemiluminescent substrate-containing agent.

The five estradiol immunodetection reagents are prepared at first, 60 estradiol immunodetection reagents are respectively prepared, the kit is put into a reagent bin of a Chenna biological FC-813 instrument, and 5 estradiol immunodetection reagents are used for respectively testing 60 clinical serum samples. Simultaneously 60 clinical serum samples are detected in Roche by using Roche estradiol detection kit (electrochemical luminescence method)

And (4) measuring the concentration of the estradiol on an E411 electrochemical luminescence full-automatic immunoassay analyzer.

The test results of the immunodetection reagents of each example were compared with the test results of the Roche estradiol assay kit (electrochemiluminescence method) of Roche diagnostics products, Inc. (Shanghai) as shown in FIGS. 1 to 5, wherein the abscissa is the test result (unit: pg/mL) of the Roche estradiol assay kit and the ordinate is the test result (unit: pg/mL) of the immunodetection kit using the different sample-releasing agents of this example. As can be seen from the experimental results shown in the figure, this example combines two methods of competitive substitution and reducing the binding force of estradiol and sex hormone binding protein, so that estradiol can be rapidly released.

As can be seen from FIGS. 1,2, 3,4 and 5, the correlation coefficient R between the detection result of the immunoassay kit without the releasing agent and the detection result of the Roche estradiol detection kit²0.6494 only, the correlation coefficient R between the detection result of the immunoassay kit containing the sample releasing agent 1 and the detection result of the Roche estradiol detection kit²Up to 0.9752, the correlation coefficient R between the detection result of the immunoassay kit containing the sample releasing agent 2 and the detection result of the Roche estradiol detection kit²Up to 0.9723 detection of an immunoassay kit containing sample Release agent 3Correlation coefficient R between detection result and detection result of Roche estradiol detection kit²Up to 0.9755, the correlation coefficient R between the detection result of the immunoassay kit containing the sample releasing agent 4 and the detection result of the Roche estradiol detection kit²Up to 0.9826.

The results of fig. 2, fig. 3, fig. 4, and fig. 5 illustrate that when the quantitative detection kit for enzymatic immunochemiluminescence of nanoparticles prepared in this example is used to detect estradiol, it is not necessary to mix estradiol releasing agent with sample in advance, and after standing for a certain period of time, add estradiol magnetic separation reagent, biotinylated estradiol antibody reagent, alkaline phosphatase labeled estradiol antigen reagent, and reagent containing enzymatic chemiluminescent substrate, but rather, the sample to be detected is mixed with estradiol releasing agent, estradiol magnetic separation reagent, biotinylated estradiol antibody reagent, and alkaline phosphatase labeled estradiol antigen reagent, and after the completion of the immunoreaction, then add reagent containing enzymatic chemiluminescent substrate, thereby effectively simplifying experimental operation, increasing detection efficiency, and making estradiol in the sample have excellent release effect.

As can be seen from FIG. 5, after a non-hormonal substance 3, 4-Divanilyltetrahydrofuran (DVT) is applied as a releasing agent in the competitive substitution reagent of the sample releasing agent 4, the correlation coefficient with the Roche estradiol detection kit is higher than that of the immunoassay kit containing other releasing agents, and the correlation coefficient R is higher than that of the immunoassay kit containing other releasing agents²0.9826 is reached, indicating that 3, 4-divanilyltetrahydrofuran has an excellent promoting effect on the release of estradiol.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims

1. An estradiol releasing agent that is capable of affecting the binding of estradiol to sex hormone binding protein, thereby releasing estradiol, the estradiol releasing agent comprising at least one of an agent for competitively binding to sex hormone binding protein, an agent for reducing the binding of estradiol to sex hormone binding protein;

the agent for competitive binding to sex hormone binding protein contains at least one of dihydrotestosterone, danazol, mesterone, testosterone, cortisol, 3, 4-divanilyltetrahydrofuran, and levonorgestrel;

the reagent for reducing the binding force of estradiol and sex hormone binding protein contains at least one of heparin sodium, 8-aniline-1-naphthalene sulfonic acid, 8-aniline-1-naphthalene sulfonate and sodium trichloroacetate.

2. The estradiol releasing agent of claim 1, comprising at least one of the following components in the following concentrations: 0.1-0.5mg/mL dihydrotestosterone, 0.05-0.3mg/mL danazol, 0.02-0.25mg/mL mesterone, 0.01-0.2mg/mL testosterone, 10-50mg/mL heparin sodium, 0.02-0.1mg/mL cortisol, 5-20mg/mL 8-aniline-1-naphthalenesulfonic acid, 1-20mg/mL sodium trichloroacetate, 0.05-0.25mg/mL 3, 4-divanilyltetrahydrofuran, 0.2-0.4mg/mL levonorgestrel.

3. The estradiol releasing agent of claim 2, comprising the following components in concentrations: 0.05-0.3mg/mL danazol and 1-20mg/mL sodium trichloroacetate;

optionally, the following concentrations of components are included: 0.1-0.5mg/mL dihydrotestosterone and 1-20mg/mL sodium trichloroacetate;

optionally, the following concentrations of components are included: 0.02-0.1mg/mL of cortisol, 0.02-0.25mg/mL of mesterone and 5-20mg/mL of 8-aniline-1-naphthalenesulfonic acid;

optionally, the following concentrations of components are included: 0.05-0.3mg/mL danazol, 0.05-0.25mg/mL 3, 4-divanilyltetrahydrofuran, 5-20mg/mL 8-aniline-1-naphthalenesulfonic acid.

4. The estradiol releasing agent of any one of claims 1 to 3, further comprising a buffer;

optionally, the buffer solution is selected from at least one of phosphate buffer solution, 2-morpholine ethanesulfonic acid buffer solution, Tris-HCl buffer solution, citrate buffer solution, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution, glycine-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution and 4-hydroxyethyl piperazine ethanesulfonic acid.

5. The estradiol releasing agent of any one of claims 1 to 3, further comprising a solvent;

optionally, the solvent is selected from at least one of organic solvent and inorganic solvent;

optionally, the solvent is selected from at least one of dimethyl sulfoxide, dimethylformamide, and water.

6. A test kit comprising the estradiol releasing agent according to any one of claims 1 to 5.

7. The test kit of claim 6, further comprising a estradiol magnetic separation reagent, a estradiol antibody reagent, a estradiol antigen reagent, an enzymatic chemiluminescent substrate reagent.

8. The detection kit of claim 7, wherein the magnetic microparticles in the estradiol magnetic separation reagent are selected from at least one of streptavidin superparamagnetic nanoparticles, carboxylated magnetic nanoparticles, tosyl magnetic nanoparticles;

optionally, the estradiol antibody in the estradiol antibody reagent, the estradiol antigen in the estradiol antigen reagent is selected from any one of the following combinations:

1) biotin-labeled estradiol antibodies, enzyme-labeled estradiol antigens;

2) biotin-labeled estradiol antigens, enzyme-labeled estradiol antibodies;

optionally, the enzyme is selected from at least one of alkaline phosphatase and horseradish peroxidase;

optionally, the enzymatic chemiluminescent substrate is selected from at least one of 3-aminophthalic hydrazide, 1, 2-dioxane derivatives, (4-chlorobenzenethiol) (10-methyl-9, 10-acridanemethylene) disodium phosphate;

optionally, the 1, 2-dioxetane derivative is selected from at least one of AMPPD, CSPD, CDP-STAR, ADP-STAR;

optionally, the concentration of the magnetic microspheres in the estradiol magnetic separation reagent is 0.01-0.05 wt%;

optionally, the concentration of estradiol antibody in the estradiol antibody reagent is 2-10 μ g/mL;

optionally, the concentration of estradiol antigen in the estradiol antigen agent is 0.5-4.0 μ g/mL;

optionally, the concentration of the enzymatic chemiluminescent substrate in the enzymatic chemiluminescent substrate reagent is 0.02 to 4 mmol/L.

9. The method for detecting estradiol by using the detection kit according to any one of claims 6 to 8, wherein a sample to be detected, the estradiol releasing reagent, the estradiol magnetic separation reagent, the estradiol antibody reagent and the estradiol antigen reagent are mixed and subjected to an immunoreaction, and the resulting mixture contains at least one of magnetic microspheres at a concentration of 0.0025 to 0.0125 wt%, an estradiol antibody at a concentration of 0.5 to 2.5 μ g/mL, an enzyme-labeled estradiol antigen at a concentration of 0.125 to 1.0 μ g/mL, and estradiol releasing agents at the following concentrations:

0.025-0.125mg/mL dihydrotestosterone, 0.0125-0.075mg/mL danazol, 0.005-0.0625mg/mL mesterone, 0.0025-0.05mg/mL testosterone, 2.5-4.0mg/mL heparin sodium, 0.005-0.025mg/mL cortisol, 1.25-5mg/mL 8-aniline-1-naphthalene sulfonic acid, 0.25-5mg/mL sodium trichloroacetate, 0.0125-0.0625mg/mL 3, 4-divanilyltetrahydrofuran, 0.05-0.1mg/mL levonorgestrel;

optionally, after completion of the immunological reaction, adding an enzymatic chemiluminescent substrate reagent to obtain a reagent useful for estradiol detection, wherein the concentration of the enzymatic chemiluminescent substrate in the reagent is 0.01-2.0 g/L.

10. Use of the estradiol releasing agent according to any one of claims 1 to 5 or the test kit according to any one of claims 6 to 8 for the detection of estradiol.