Estrogen sample treatment agents, their use in estrogen detection and detection methods

Abstract

The present application provides an estradiol sample treatment agent and its application in estradiol detection and a detection method, the method comprising: a displacement component for releasing estradiol in a bound state in a sample into a free state; a metal ion. The estradiol sample treatment agent of the present application can quickly and efficiently release the bound estradiol into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, being suitable for popularization and application, and having high application value.

Description

Technical Field

[0001] This invention relates to the field of biology. Specifically, this invention relates to estradiol sample preparation agents and their application and detection methods in estradiol detection. Background Technology

[0002] Estradiol (E2) is a steroid hormone with a basic steroidal structure, namely cyclopentanoperhydrophenanthrene, composed of three cyclohexanes and one cyclopentane fused together. Estradiol is the most abundant and biologically active estrogen, playing a crucial role in the reproductive system, immune responses, and gene expression in mammals. In the blood, E2 is an important diagnostic marker in various clinical conditions in both men and women.

[0003] 40-60% of E2 binds to sex hormone-binding globulin (SGH), the remainder binds nonspecifically to albumin, and only a small fraction of E2 exists in a free state. In clinical diagnosis, the total amount of E2 is usually measured, thus requiring the release of E2 bound to SGH and albumin into a free state. Current techniques typically use substances with stronger affinity for SGH, such as danazol and testosterone, to replace E2 in the sample. However, the replacement effect is not ideal, easily leading to inaccurate test results. Therefore, there is an urgent need for a sample processing agent that can fully and efficiently release bound E2. Summary of the Invention

[0004] This invention aims to at least partially address the technical problems existing in the prior art. To this end, this invention proposes an estradiol sample processing agent, an estradiol immunoassay system, and their application in the detection of estradiol, as well as a method for detecting estradiol samples using the estradiol immunoassay system. The estradiol sample processing agent of this invention can rapidly and efficiently release bound estradiol into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, and making it suitable for widespread application with high application value.

[0005] In one aspect, the present invention provides an estradiol sample processing agent. According to an embodiment of the present invention, the estradiol sample processing agent comprises: a displacement component for releasing bound estradiol in the sample into a free state; and metal ions. The estradiol sample processing agent according to the embodiments of the present invention, through the synergistic effect of the displacement component and the metal ions, further enhances the displacement effect, enabling the rapid and efficient release of bound estradiol in the sample into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, and demonstrating high application value.

[0006] According to embodiments of the present invention, the above-mentioned estradiol sample treatment agent may also have the following additional technical features:

[0007] According to embodiments of the present invention, the replacement component includes at least one of the following: testosterone, danazol, methyldihydrotestosterone, dihydrotestosterone, levonorgestrel, cortisol, diethylstilbestrol, divanillyltetrahydrofuran, 8-aniline-1-naphthalenesulfonic acid, and sodium trichloroacetate.

[0008] According to an embodiment of the present invention, the concentration of the replacement component is 0.1 to 0.5 mmol / L.

[0009] According to embodiments of the present invention, the metal ions include zinc ions and / or calcium ions.

[0010] According to an embodiment of the present invention, the concentration of the metal ion is 0.05 to 0.7 mmol / L.

[0011] According to an embodiment of the present invention, the estradiol sample processing agent further comprises: a base component; the base component includes at least one of the following: a buffer, a protein protectant, and a preservative.

[0012] According to embodiments of the present invention, the buffer solution includes at least one of phosphate buffer, acetate buffer, citrate buffer, morpholine ethanesulfonate buffer, borate buffer, Tris hydrochloric acid buffer, acetate-sodium acetate buffer, and carbonate buffer.

[0013] According to embodiments of the present invention, the protein protectant comprises one or more of bovine serum albumin or casein.

[0014] According to embodiments of the present invention, the preservative includes one or more of sodium azide, proclin, and thimerosal, thereby improving the stability of the system without interfering with the displacement reaction.

[0015] According to an embodiment of the present invention, the pH value of the estradiol sample treatment agent is 5.5 to 6.5.

[0016] In another aspect, the present invention provides an estradiol immunoassay system. According to an embodiment of the invention, the system comprises the aforementioned estradiol sample processing agent. Thus, the estradiol immunoassay system of the present invention can effectively release bound estradiol in an estradiol sample into free estradiol, thereby facilitating estradiol detection and improving detection accuracy.

[0017] According to an embodiment of the present invention, the estradiol immunoassay system further comprises at least one of the following: a first antibody, a second antibody, and an excitation substrate; the first antibody is specifically capable of binding estradiol; the second antibody is specifically capable of binding the new conformation formed after estradiol binds to the first antibody.

[0018] According to an embodiment of the present invention, the first antibody is coated on a solid support, and the second antibody is labeled with a signal generator.

[0019] According to an embodiment of the present invention, the second antibody is coated on a solid support, and the first antibody is labeled with a signal generator;

[0020] According to embodiments of the present invention, the signal generator includes isoluminol and its derivatives, luminol, acridine ester, ruthenium tripyridine, horseradish peroxidase, and alkaline phosphatase.

[0021] According to an embodiment of the present invention, the estradiol immunoassay system is provided in the form of a reagent kit.

[0022] In another aspect, the present invention proposes the application of the aforementioned estradiol sample processing agent or the aforementioned estradiol immunoassay system in the detection of estradiol. As mentioned above, the aforementioned estradiol sample processing agent or estradiol immunoassay system can rapidly and efficiently release bound estradiol into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, and demonstrating high application value.

[0023] According to an embodiment of the present invention, the estradiol sample is selected from bodily fluid samples.

[0024] According to an embodiment of the present invention, the body fluid sample includes serum and plasma.

[0025] According to an embodiment of the present invention, the blood collection tube for the plasma sample is selected from any one of sodium citrate tubes, sodium heparin tubes, and lithium heparin.

[0026] In addition, this invention proposes the application of the aforementioned estradiol sample processing agent or the aforementioned estradiol immunoassay system in the preparation of reagent kits.

[0027] In another aspect, the present invention provides a method for detecting estradiol using the aforementioned estradiol immunoassay system. According to an embodiment of the present invention, the method includes: reacting the estradiol sample processing agent with an estradiol sample, a solid-phase carrier coated with a first antibody, and a second antibody labeled with a signal generator; or reacting the estradiol sample processing agent with an estradiol sample, a first antibody labeled with a signal generator, and a solid-phase carrier coated with a second antibody to obtain an estradiol-first antibody-second antibody complex; mixing the estradiol-first antibody-second antibody complex with an excitation substrate and detecting the emitted relative light intensity; and analyzing the estradiol in the estradiol sample based on the relative light intensity. Therefore, the method of the present invention can accurately detect estradiol, is simple and efficient, and is suitable for widespread application.

[0028] According to an embodiment of the present invention, the estradiol sample is selected from bodily fluid samples.

[0029] According to an embodiment of the present invention, the body fluid sample includes serum and plasma.

[0030] According to an embodiment of the present invention, the blood collection tube for the plasma sample is selected from any one of sodium citrate tubes, sodium heparin tubes, and lithium heparin.

[0031] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0033] Figure 1 The correlation analysis diagram of chemiluminescence immunoassay sandwich detection and mass spectrometry detection according to an embodiment of the present invention is shown. Detailed Implementation

[0034] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0035] This invention proposes the application of estradiol sample processing agents, estradiol immunoassay systems, and the application of estradiol sample processing agents or estradiol immunoassay systems in the detection of estradiol and in the preparation of reagent kits, as well as methods for detecting estradiol using estradiol immunoassay systems. These will be described in detail below.

[0036] estradiol sample preparation agent

[0037] In one aspect of the invention, an estradiol sample treatment agent is provided. According to an embodiment of the invention, the estradiol sample treatment agent comprises: a displacement component for releasing bound estradiol in the sample into a free state; and metal ions.

[0038] The estradiol sample treatment agent according to embodiments of the present invention, through the synergistic effect of the replacement components and metal ions, further enhances the replacement effect, enabling the rapid and efficient release of bound estradiol in the sample into free estradiol.

[0039] Specifically, although some estradiol in an estradiol sample is bound to albumin, albumin has a weak binding affinity, so this portion of E2 can be easily released once a treatment agent is added. Therefore, in the field of in vitro diagnostics, research is usually focused on how to release E2 that is bound to sex hormone-binding proteins with high affinity.

[0040] By adding a replacement component, which has a stronger affinity for sex hormone-binding proteins than estradiol, bound estradiol is displaced to obtain free estradiol. However, using only the aforementioned replacement component cannot achieve a truly ideal replacement effect. Therefore, the inventors creatively added metal ions to the sample processing agent, finding that this significantly improved the replacement rate. The inventors hypothesize that the metal ions in the sample processing agent may interfere with the binding force between E2 and sex hormone-binding proteins. Since the replacement and binding process is dynamic, the displaced E2 still retains binding force with the sex hormone-binding protein, and a small portion may re-enter the bound state before detection. When metal ions are present, their interference with the E2 binding force not only assists the replacement component in releasing free E2 but also prevents the free E2 from re-binding to the protein, thus working synergistically with the replacement component to achieve a thorough replacement effect.

[0041] Therefore, the estradiol sample processing agent of the present invention can rapidly and efficiently release bound estradiol into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, and having high application value.

[0042] According to embodiments of the present invention, the replacement component comprises at least one of the following: testosterone, danazol, methyldihydrotestosterone, dihydrotestosterone, levonorgestrel, cortisol, diethylstilbestrol, divanillyltetrahydrofuran, 8-aniline-1-naphthalenesulfonic acid, and sodium trichloroacetate. Both the above-mentioned replacement components and estradiol bind to sex hormone-binding proteins, and the binding sites of sex hormone-binding proteins are unique. The affinity of the replacement components for sex hormone-binding proteins is higher than that for estradiol, allowing them to competitively bind to sex hormone-binding proteins and release estradiol. Methyldihydrotestosterone is particularly effective.

[0043] According to embodiments of the present invention, the concentration of the replacement component is 0.1–0.5 mmol / L. In some embodiments, the concentration of the replacement component is 0.1 mmol / L, 0.15 mmol / L, 0.2 mmol / L, 0.25 mmol / L, 0.3 mmol / L, 0.35 mmol / L, 0.4 mmol / L, 0.45 mmol / L, 0.5 mmol / L, or any two values ​​as a range between two endpoints. This allows for sufficient replacement of estradiol bound to sex hormone-binding globulin and albumin.

[0044] According to embodiments of the present invention, the metal ions include zinc ions and / or calcium ions. Using zinc ions and / or calcium ions can further improve the displacement efficiency, effectively releasing bound estradiol.

[0045] According to embodiments of the present invention, the concentration of the metal ion is 0.05–0.7 mmol / L. In some embodiments, the concentration of the metal ion is 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, or any two values ​​as a range between two endpoints. In some embodiments, the concentration of the metal ion is 0.1–0.5 ng / mL. This can further improve the displacement efficiency.

[0046] According to embodiments of the present invention, the estradiol sample processing agent further comprises: a base component; the base component includes at least one of the following: a buffer solution, a protein protectant, and a preservative. The buffer solution provides a suitable system environment for the displacement reaction, the protein protectant maintains the stability of proteins in the reaction system, and the preservative improves the stability of the system, facilitating storage and transportation.

[0047] According to embodiments of the present invention, the buffer solution comprises at least one selected from phosphate buffer, acetate buffer, citrate buffer, morpholine ethanesulfonate buffer, borate buffer, Tris hydrochloride buffer, acetate-sodium acetate buffer, and carbonate buffer. This facilitates the occurrence of the displacement reaction.

[0048] According to embodiments of the present invention, the protein protectant comprises one or more of bovine serum albumin or casein.

[0049] According to embodiments of the present invention, the preservative includes one or more of sodium azide, proclin, and thimerosal. This improves the stability of the system without interfering with the displacement reaction.

[0050] According to embodiments of the present invention, the pH value of the estradiol sample treatment agent is 5.5 to 6.5. In some embodiments, the pH value of the estradiol sample treatment agent is 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, or any two values ​​as a range between two endpoints. This facilitates the occurrence of the displacement reaction.

[0051] Estradiol Immunoassay System

[0052] In another aspect, the present invention provides an estradiol immunoassay system. According to an embodiment of the present invention, the system includes the aforementioned estradiol sample processing agent. Thus, the estradiol immunoassay system of the present invention can effectively release bound estradiol in an estradiol sample into free estradiol, thereby facilitating estradiol detection and improving detection accuracy. Specifically, the above-mentioned estradiol immunoassay system can be provided in the form of a kit; that is, the present invention provides an estradiol detection kit comprising the aforementioned estradiol immunoassay system.

[0053] According to an embodiment of the present invention, the estradiol immunoassay system further comprises at least one of the following: a first antibody, a second antibody, and an excitation substrate; the first antibody is specifically capable of binding estradiol; the second antibody is specifically capable of binding the new conformation formed after estradiol binds to the first antibody.

[0054] According to an embodiment of the present invention, the first antibody is coated on a solid support, and the second antibody is labeled with a signal generator.

[0055] According to another embodiment of the present invention, the second antibody is coated on a solid support, and the first antibody is labeled with a signal generator.

[0056] According to embodiments of the present invention, the signal generator includes isoluminol and its derivatives, luminol, acridine ester, ruthenium tripyridine, horseradish peroxidase, and alkaline phosphatase.

[0057] According to an embodiment of the present invention, the basic structure of the isoluminol derivative is as follows: Wherein, R1 is selected from C2H5, C3H7, or C4H9, and R2 is selected from NH2-(CH2)4, NH2-(CH2)6, NH2-(CH2)8, or NH2-(CH2). 10 .

[0058] The aforementioned luminescent marker is a compound that participates in energy transfer in the luminescent reaction and ultimately releases energy in the form of photons. This compound can be catalyzed by a catalyst and oxidized by an oxidant to form an excited-state intermediate. When this excited-state intermediate returns to the stable ground state, it emits a photon (hM).

[0059] According to an embodiment of the present invention, the isoluminol derivative is selected from N-(4-aminobutyl)-N-ethylisoluminol.

[0060] According to embodiments of the present invention, the excitation substrate is selected from NaOH and H2O2. When isoluminol or isoluminol derivatives react with hydroxides, a bis-ion is generated, which can be oxidized by oxygen released from the decomposition of hydrogen peroxide to generate a very unstable peroxide, which immediately decomposes to release nitrogen gas, generating excited-state 3-aminophthalic acid.

[0061] This detection system effectively avoids the risk of cross-reactions caused by the structural similarity between the replacement component and estradiol. Because it employs a sandwich method, even if the first antibody non-specifically binds to the replacement component, the high specificity of the second antibody (which only binds to the new conformational epitope formed by the specific binding of the first antibody and estradiol) ensures that the final test complex is a ternary immune complex of estradiol-first antibody-second antibody. This efficiently replaces estradiol with the sample processing agent while avoiding inaccurate test results due to potential cross-reactions.

[0062] application

[0063] In another aspect, the present invention proposes the application of the aforementioned estradiol sample processing agent or the aforementioned estradiol immunoassay system in the detection of estradiol. As mentioned above, the aforementioned estradiol sample processing agent or estradiol immunoassay system can rapidly and efficiently release bound estradiol into free estradiol, thereby facilitating estradiol detection, improving detection accuracy, and demonstrating high application value. Specifically, the direct purpose of detecting estradiol samples is not to obtain diagnostic results or health status, but rather to provide information as an intermediate result.

[0064] According to an embodiment of the present invention, the estradiol sample is selected from bodily fluid samples.

[0065] According to an embodiment of the present invention, the body fluid sample includes serum and plasma.

[0066] According to an embodiment of the present invention, the blood collection tube for the plasma sample is selected from any one of sodium citrate tubes, sodium heparin tubes, and lithium heparin. EDTA plasma tubes are not suitable for this invention because they are treated with metal chelating agents EDTA-2K or EDTA-2Na, which can react with metal ions in the replacement agent and affect the replacement effect.

[0067] In addition, this invention proposes the application of the aforementioned estradiol sample processing agent or the aforementioned estradiol immunoassay system in the preparation of reagent kits.

[0068] method

[0069] In another aspect, the present invention provides a method for detecting estradiol using the aforementioned estradiol immunoassay system. According to an embodiment of the present invention, the method includes: reacting the estradiol sample processing agent with an estradiol sample, a solid-phase carrier coated with a first antibody, and a second antibody labeled with a signal generator; or reacting the estradiol sample processing agent with an estradiol sample, a first antibody labeled with a signal generator, and a solid-phase carrier coated with a second antibody to obtain an estradiol-first antibody-second antibody complex; mixing the estradiol-first antibody-second antibody complex with an excitation substrate and detecting the emitted relative light intensity; and analyzing the estradiol in the estradiol sample based on the relative light intensity. Therefore, the method of the present invention can accurately detect estradiol, is simple and efficient, and is suitable for widespread application.

[0070] According to an embodiment of the present invention, an estradiol sample treatment agent is mixed and incubated with an estradiol sample so that the replacement component and metal ions release the bound estradiol in the sample to obtain a test solution containing free estradiol.

[0071] It should be noted that in the detection process of the present invention, the test solution, the solid-phase carrier coated with the first antibody, and the second antibody labeled with the signal generator can react in one step or in steps to generate an estradiol-first antibody-second antibody complex.

[0072] In some embodiments, the test solution, a solid-phase carrier coated with a first antibody, and a second antibody labeled with a signal generator are mixed and incubated, then washed to obtain an estradiol-first antibody-second antibody complex.

[0073] In other embodiments, the test solution, a first antibody labeled with a signal generator, and a solid-phase carrier coated with a second antibody are mixed, incubated, and washed to obtain an estradiol-first antibody-second antibody complex.

[0074] According to an embodiment of the present invention, the estradiol sample is selected from bodily fluid samples.

[0075] According to an embodiment of the present invention, the body fluid sample includes serum and plasma.

[0076] According to an embodiment of the present invention, the blood collection tube for the plasma sample is selected from any one of sodium citrate tubes, sodium heparin tubes, and lithium heparin.

[0077] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Examples and Comparative Examples

[0079] 1. Prepare sample processing reagents

[0080] Weigh 19.5 g of morpholine ethanesulfonic acid (MES) and dissolve it in 500 mL of DEPC water. Add the replacement components and metal ions according to the concentrations in Table 1, where zinc ions are provided by ZnSO4 aqueous solution and calcium ions are provided by CaCl2 aqueous solution. Then add 0.5 wt% bovine serum albumin (BSA) and 0.2 wt% sodium azide. Adjust the pH with 0.5 M NaOH solution and store the solution at 4 °C for later use.

[0081] The types and concentrations of the replacement components and metal ions, as well as the pH value of the sample treatment agent, are shown in Table 1.

[0082] Table 1 Sample treatment agents

[0083]

[0084]

[0085] 2. Testing

[0086] (1) The free estradiol obtained from external purchase was divided into two portions: 1) One portion was mixed with 600 nM natural sex hormone binding protein at a volume ratio of 1:1 and reacted at 37°C for 40 min to allow the free E2 to bind with the sex hormone binding protein to form a bound state, thus obtaining a bound estradiol sample; 2) The other portion was mixed with PBS buffer at a volume ratio of 1:1 to obtain a free estradiol sample.

[0087] (2) Chemiluminescence immunoassay was selected. The MAGLUMI 4000P chemiluminescence immunoassay analyzer and the estradiol sandwich assay kit, both manufactured by New Industries, were used to detect the different test solutions obtained in the previous step. The specific detection steps are as follows:

[0088] (2-1) The test solution and the first antibody that specifically binds to E2 coated magnetic microspheres and the prepared sample processing reagents are mixed together at a volume ratio of 1:1:4 and incubated for 10 min. The estradiol in the test solution binds to the first antibody coated magnetic microspheres to form an immune complex. After incubation, unbound substances are removed by magnetic separation and washing.

[0089] (2-2) A second antibody labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was added to specifically bind E2 and form a new conformation with the first antibody. The reaction was carried out for 10 min, and unbound substances were removed by magnetic separation washing after incubation. Finally, substrate solution (containing NaOH and H2O2) for the fully automated immunoassay system was added to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) measured by the photomultiplier tube was proportional to the E2 concentration in the test solution. The relative deviation of the estradiol concentration detection results in bound estradiol samples and free estradiol samples treated with the same sample treatment agent was calculated. The lower the deviation, the higher the replacement effect. The results are shown in Table 2.

[0090] Table 2 Analysis of Relative Deviation Results

[0091] Group deviation Group deviation Comparative Example 1 11.4％ Example 5 9.10％ Comparative Example 2 24.8％ Example 6 10.10％ Comparative Example 3 8.1％ Example 7 11.60％ Comparative Example 4 10.5％ Example 8 12.10％ Comparative Example 5 15.1％ Example 9 15.10％ Comparative Example 6 14.8％ Example 10 16.10％ Comparative Example 7 19.9％ Example 11 4.40％ Comparative Example 8 20.8％ Example 12 7.00％ Comparative Example 9 21.3％ Example 13 5.70％ Comparative Example 10 26.5％ Example 14 4.40％ Example 1 5.70％ Example 15 2.0％ Example 2 15.20％ Example 16 1.0％ Example 3 0.80％ Example 17 1.2％ Example 4 5.10％ Example 18 1.9％

[0092] The data above show that the displacement effect of each sample treatment agent was significantly improved to varying degrees after the addition of metal ions. In Example 1, the deviation from the free test solution was only 5.70%, representing a two-fold improvement in displacement capacity compared to Comparative Example 1. In particular, in Example 3, when zinc ions at a concentration of 0.2 mmol / L were added to the 0.15 mM / L methyldihydrotestosterone (MDT) as the displacement component, the deviation from the free test solution was only 0.80%, and the displacement capacity was approximately ten times higher than that of Comparative Example 3, achieving unexpected technical results.

[0093] 3. Correlation with mass spectrometry results

[0094] Sixty-six plasma samples (20 males, 20 females, 20 children, and 6 pregnant women) from the Nileke County Maternal and Child Health Hospital were selected. The samples were treated with the sample processing agent described in Example 3 and detected using the aforementioned chemiluminescence immunoassay. The results were compared with those obtained by mass spectrometry. The mass spectrometry detection steps are as follows:

[0095] Mass spectrometry detection method: Sample pretreatment process:

[0096] ① Accurately weigh the sample to be analyzed (approximately 0.05-1.0 mL) into a 5 mL centrifuge tube, add the internal standard working solution (500 pg / g labeled 17β-estradiol-[13C3][17β-estradiol-13C3](E2-13C3)), record the mass of the sample and internal standard working solution accurately weighed by the balance, so that the mass ratio of the target analyte to the internal standard is approximately 1:1, add ultrapure water to approximately 1 mL of the sample solution, and equilibrate at room temperature for 1 hour;

[0097] ② Add 1.5 mL of extraction solution (hexane / ethyl acetate = 1:1 v / v) to the sample tube, mix thoroughly using a vortex mixer, then mix in a roller mixer for 10 min, centrifuge at 5500 rpm for 10 min, transfer 1 mL of supernatant and dry at 45 °C; continue to add 1.5 mL of extraction solution to the sample tube, and repeat the mixing, centrifugation, transfer and nitrogen blowing steps;

[0098] ③ After drying, add 200 μL of reconstitution solution, place in a roller mixer and mix for 10 min, filter and then test on the instrument.

[0099] Test index description: y = ax + b, where a is the slope, representing the overall deviation (1) between the chemiluminescence immunoassay sandwich method and the mass spectrometry detection results; the closer a slope is to 1, the smaller the deviation. b is the intercept, representing the overall deviation (2) between the chemiluminescence immunoassay sandwich method and the mass spectrometry detection results; the closer a intercept is to 0, the smaller the deviation. Results are shown below. Figure 1 .

[0100] The results showed that, when testing the reagent samples, the sample processing agent provided in Example 3 achieved a correlation of 0.9989 with mass spectrometry after sample processing. This further demonstrates that using the replacement component and zinc ions as E2 release agents can fully release protein-bound E2 in the sample. That is, when metal ions such as zinc ions and / or calcium ions are present, even in complex blood samples, they can still interfere with the binding force of E2, assist the replacement component in releasing free E2, and prevent the free E2 from rebinding to the protein. They work synergistically with the replacement component to achieve a full replacement effect.

[0101] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An estradiol sample treatment agent, characterized in that, include: A displacement component, wherein the displacement component is used to release estradiol in the sample from a bound state into a free state. The replacement component is selected from at least one of the following: testosterone, methyldihydrotestosterone, dihydrotestosterone, levonorgestrel, cortisol, diethylstilbestrol, and divanillyltetrahydrofuran; Compounds containing metal ions, including zinc sulfate and / or calcium chloride; The concentration of the replacement component is 0.15~0.45 mmol / L; The concentration of the metal ions is 0.1~0.5 mmol / L; The pH value of the estradiol sample treatment agent is 5.5~6.

5.

2. The estradiol sample treatment agent according to claim 1, characterized in that, Further includes: Basic components; The basic components include at least one of the following: buffer solution, protein protectant, and preservative; Optionally, the buffer solution comprises at least one of phosphate buffer, acetate buffer, citrate buffer, morpholine ethanesulfonic acid buffer, borate buffer, Tris hydrochloric acid buffer, acetate-sodium acetate buffer, and carbonate buffer. Optionally, the protein protectant includes one or more of bovine serum albumin or casein; Optionally, the preservative includes one or more of sodium azide, proclin, and thimerosal.

3. An estradiol immunoassay system, characterized in that, include: The estradiol sample treatment agent according to claim 1 or 2.

4. The estradiol immunoassay system according to claim 3, characterized in that, Further comprising at least one of the following: a primary antibody, a secondary antibody, and an activation substrate; The first antibody can specifically bind to estradiol; The second antibody can specifically bind to the new conformation formed after estradiol binds to the first antibody.

5. The estradiol immunoassay system according to claim 4, characterized in that, The first antibody is coated on a solid support, and the second antibody is labeled with a signal generator. Optionally, the second antibody is coated on a solid support, and the first antibody is labeled with a signal generator.

6. The estradiol immunoassay system according to claim 5, characterized in that, The signal generators include isoluminol, luminol, acridine ester, terpyridine ruthenium, horseradish peroxidase, or alkaline phosphatase.

7. The estradiol immunoassay system according to any one of claims 3-6, characterized in that, The estradiol immunoassay system is provided in the form of a reagent kit.

8. The use of the estradiol sample processing agent according to claim 1 or 2 or the estradiol immunoassay system according to any one of claims 3-7 in the detection of estradiol.

9. The application according to claim 8, characterized in that, The estradiol sample was selected from body fluid samples.

10. The application according to claim 9, characterized in that, The body fluid sample includes serum, plasma, or whole blood.

11. The use of the estradiol sample processing agent of claim 1 or 2 or the estradiol immunoassay system of any one of claims 3-7 in the preparation of a reagent kit.

12. A method for detecting estradiol using the estradiol immunoassay system according to any one of claims 3-7, characterized in that, include: The estradiol sample processing agent is reacted with an estradiol sample, a solid-phase carrier coated with a first antibody, and a second antibody labeled with a signal generator; or, the estradiol sample processing agent is reacted with an estradiol sample, a first antibody labeled with a signal generator, and a solid-phase carrier coated with a second antibody to obtain an estradiol-first antibody-second antibody complex. The estradiol-first antibody-second antibody complex was mixed with an excitation substrate, and the relative intensity of the emitted light was detected. Based on the relative light intensity, the estradiol in the estradiol sample was analyzed.

13. The method according to claim 12, characterized in that, The estradiol sample was selected from body fluid samples.

14. The method according to claim 13, characterized in that, The body fluid samples include serum and plasma.

15. The method according to claim 14, characterized in that, The plasma collection tubes are selected from any one of sodium citrate tubes, sodium heparin tubes, and lithium heparin tubes.

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