Application of a dissociating agent in an estradiol detection kit

Abstract

The present invention discloses the use of a dissociating agent in an estradiol detection kit, belonging to the field of biotechnology. The concentration of perfluorooctanoic acid in the dissociating agent is 0.01-1 mg / L. The dissociating agent of the present invention allows the dissociation process and the immune process to occur simultaneously, and the detection results of the immune detection kit are highly correlated with the detection results of the Roche estradiol detection kit, which is superior to other dissociating agents. It can quickly and effectively dissociate estradiol from albumin and sex hormone binding protein, thereby improving the sensitivity of estradiol detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a dissociating agent in an estradiol detection kit. Background Art

[0002] Estradiol is the most important female hormone, promoting and regulating the normal development of female sexual organs and secondary sexual characteristics. It participates in the metabolism of proteins, sugars, lipids, water, electrolytes, calcium, and phosphorus, regulates vascular smooth muscle and endothelial cells, lowers cholesterol, and significantly promotes growth and improves production performance. Furthermore, estradiol prevents osteoporosis, reduces the risk of cardiovascular disease, and regulates the hypothalamus. Estradiol concentrations in the human body are extremely low, but its physiological activity is highly significant. Monitoring its normal level is of great clinical significance.

[0003] Current methods for detecting estradiol include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). These methods offer the advantages of high sensitivity and accuracy. However, these methods require the separation and concentration of estradiol prior to detection, necessitating complex pretreatment procedures. Existing immunoassay reagents require sample processing before the estradiol is released from binding proteins and then undergo an immune reaction, adding an additional step before the immune response.

[0004] Chinese patent CN113504379A discloses a sex hormone dissociator and detection kit, which includes a sex hormone dissociator, a sex hormone magnetic particle reagent, and an antibody reagent. The detection of sex hormones includes the following steps: mixing a test sample with the sex hormone dissociator to form a mixed sample; mixing the mixed sample with the antibody reagent and performing a first incubation reaction; after the first incubation reaction, adding the sex hormone magnetic particle reagent to the mixed sample and performing a second incubation reaction; adding a chemiluminescent substrate solution and reading the value. The sex hormone dissociator provided in this patent has the advantages of fast dissociation speed, high efficiency, and no impact on the reagent's reaction performance, but it has multiple reaction steps and a complex reaction process.

[0005] Estradiol primarily binds to sex hormone-binding protein (SHBP) and albumin, while a small amount of free, biologically active estradiol remains constant throughout the menstrual cycle. Immunoassays for estradiol require its release from SHBP and albumin to facilitate recognition by detection antibodies. Therefore, the formulation of estradiol releasers is crucial for estradiol release, directly impacting the sensitivity and specificity of estradiol immunoassay reagents. Existing estradiol releasers struggle to fully dissociate estradiol from albumin and SHBP.

[0006] Chinese patent CN113125696A discloses a homogeneous chemiluminescent detection kit for estradiol, comprising a first receptor microsphere coupled to a first antibody, a second receptor microsphere coupled to a second antibody, and a competing antigen for the binding of estradiol to the detection antibody. The kit described in this patent utilizes antibodies with different affinities coupled to the receptor microspheres at different mass ratios, and then mixes the two receptor microspheres in an appropriate ratio. This allows the antibodies of varying affinities to selectively act depending on the concentration of the antigen being detected, thereby expanding the detection range of the kit. However, its detection sensitivity is relatively low.

[0007] The present invention uses perfluorooctanoic acid as a dissociating component, enabling simultaneous estradiol dissociation and immune response. This allows the dissociation and immune response processes to occur simultaneously, eliminating the need for a separate dissociation (sample processing) step, thereby improving estradiol detection efficiency. The dissociating agent, as a component of the estradiol acridinium ester chemiluminescent immunoassay, can rapidly and effectively dissociate estradiol from albumin and sex hormone-binding protein, significantly improving the sensitivity and specificity of the estradiol immunoassay. Summary of the Invention

[0008] The present invention aims to provide a dissociation agent for use in an estradiol detection kit. The dissociation agent of the present invention enables the dissociation process and the immune process to occur simultaneously. The detection results of the estradiol detection kit using perfluorooctanoic acid as a dissociation agent are superior to those of other dissociation agents, thereby improving the sensitivity of estradiol detection.

[0009] In order to achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0010] In one aspect, the present invention provides use of perfluorooctanoic acid in the preparation of an estradiol dissociating agent.

[0011] Preferably, the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.01-1 mg / L.

[0012] Further preferably, the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.01-0.1 mg / L.

[0013] Most preferably, the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.05 mg / L.

[0014] Preferably, the estradiol dissociating agent consists of a dissociating component and a buffer, and the dissociating component includes perfluorooctanoic acid.

[0015] Further preferably, the dissociated component is selected from perfluorooctanoic acid, or a combination of perfluorooctanoic acid and at least one of danazol, levonorgestrel, dihydrotestosterone, and mesterolone.

[0016] Still further preferably, the dissociated component is selected from perfluorooctanoic acid, or a combination of perfluorooctanoic acid and at least one of danazol, levonorgestrel, dihydrotestosterone, and mesterolone.

[0017] Most preferably, the dissociated component is perfluorooctanoic acid.

[0018] Specifically, the molecular formula of the perfluorooctanoic acid is C8HF 15 O2, CAS number is 335-67-1.

[0019] Specifically, the chemical name of the estradiol is estradiol-1,3,5(10)-triene-3,17β-diol.

[0020] Specifically, the molecular formula of dihydrotestosterone is C 19 H 30 O2, CAS number is 521-18-6.

[0021] Specifically, the molecular formula of danazol is C 22 H 27 NO2, CAS number is 17230-88-5.

[0022] Specifically, the molecular formula of the mesterolone is C 20 H 32 O2, CAS number is 521-11-9.

[0023] Specifically, the molecular formula of testosterone is C 19 H 28 O2, CAS number is 58-22-0.

[0024] Specifically, the molecular formula of the corticosteroid is C 21 H 28 O5, CAS number is 53-06-5.

[0025] Specifically, the molecular formula of the levonorgestrel is C 21 H 28 O2, CAS number is 797-63-7.

[0026] Specifically, the molecular formula of the 3,4-divanillyltetrahydrofuran is C 20 H 24 O5, CAS number is 34730-78-4.

[0027] Preferably, the buffer is selected from at least one of PB buffer, 2-morpholineethanesulfonic acid buffer, PBS buffer, Tris-HCl buffer, citrate buffer, acetic acid-sodium acetate buffer, and 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer.

[0028] Most preferably, the buffer is Tris-HCl buffer.

[0029] Specifically, the PB buffer solution includes NaH2PO4·2H2O and Na2HPO4·12H2O.

[0030] Specifically, the molecular formula of the 2-morpholineethanesulfonic acid buffer is C6H 13 NO4S, CAS number is 4432-31-9.

[0031] Specifically, the PBS buffer solution includes NaH2PO4·2H2O, Na2HPO4·12H2O and NaCl.

[0032] Specifically, the chemical name of the Tris-HCl buffer is tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0033] Specifically, the molecular formula of the citrate buffer is C 34 H 40 FNO8, CAS number is 13754-17-1.

[0034] Specifically, the molecular formula of the acetic acid-sodium acetate is C4H7NaO4.

[0035] Specifically, the molecular formula of the 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid buffer is C7H 15 NO5S, CAS number is 68399-77-9.

[0036] On the other hand, the present invention provides an estradiol dissociator, which is composed of a dissociation component and a buffer; the dissociation component includes perfluorooctanoic acid; and the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.01-1 mg / L.

[0037] Preferably, the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.01-0.1 mg / L.

[0038] Further preferably, the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.05 mg / L.

[0039] Preferably, the buffer is selected from at least one of PB buffer, 2-morpholineethanesulfonic acid buffer, PBS buffer, Tris-HCl buffer, citrate buffer, acetic acid-sodium acetate buffer, and 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer.

[0040] Further preferably, the buffer is Tris-HCl buffer.

[0041] Preferably, the dissociation component is selected from perfluorooctanoic acid, or a combination of perfluorooctanoic acid and at least one of dihydrotestosterone, danazol, mesterolone, testosterone, corticosteroids, levonorgestrel, and 3,4-divanillyltetrahydrofuran.

[0042] Further preferably, the dissociated component is selected from perfluorooctanoic acid, or a combination of perfluorooctanoic acid and at least one of danazol, levonorgestrel, dihydrotestosterone, and mesterolone.

[0043] Most preferably, the dissociated component is perfluorooctanoic acid.

[0044] In another aspect, the present invention provides use of an estradiol dissociating agent in preparing an estradiol detection kit.

[0045] Preferably, the estradiol detection kit further comprises an estradiol magnetic separation reagent, an estradiol antibody reagent, a magnetic estradiol antigen reagent, a chemiluminescent substrate reagent, and a cleaning solution.

[0046] Further preferably, the magnetic particles in the estradiol magnetic separation reagent are selected from at least one of streptavidin (Agilent SA) magnetic particles, tosyl magnetic particles, carboxyl magnetic particles, and amino magnetic particles.

[0047] More preferably, the magnetic particles in the estradiol magnetic separation reagent are Agilent SA magnetic particles.

[0048] Further preferably, the chemiluminescent substrate in the chemiluminescent substrate reagent is a VIRTUOSO homemade substrate.

[0049] Still further preferably, the concentration of the magnetic particles in the estradiol magnetic separation reagent is 1.0 mg / mL.

[0050] Still more preferably, the concentration of the estradiol antibody in the estradiol antibody reagent is 0.05 mg / L.

[0051] Still more preferably, the concentration of estradiol antigen in the estradiol antigen reagent is 0.05 mg / L.

[0052] The above-mentioned detection kit is used to detect estradiol. The sample to be tested, the dissociation agent, the magnetic particles, the estradiol antibody reagent, and the estradiol antigen reagent are mixed to perform an immune reaction. The resulting mixed solution contains a dissociation agent with a concentration of 0.05 mg / L, magnetic particles with a concentration of 1.0 mg / mL, estradiol antibodies with a concentration of 0.05 mg / L, and estradiol antigens with a concentration of 0.05 mg / L.

[0053] The beneficial effects of the present invention are:

[0054] (1) The estradiol dissociating agent of the present invention can quickly and effectively dissociate estradiol from albumin and sex hormone binding protein, thereby improving the sensitivity and specificity of the estradiol immunoassay reagent.

[0055] (2) The estradiol dissociating agent of the present invention is used as a component of the immune reagent, which allows estradiol to dissociate and undergo immune reaction simultaneously, thereby improving the detection efficiency of estradiol. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a comparison chart of Example 1 and Roche's measured values.

[0057] Figure 2 It is a comparison chart of Example 2 and Roche's measured values.

[0058] Figure 3 It is a comparison chart of Example 3 and Roche's measured values.

[0059] Figure 4 It is a comparison chart of Example 4 and Roche's measured values.

[0060] Figure 5 It is a comparison chart of comparative example 1 and Roche's measured values.

[0061] Figure 6 It is a comparison chart of comparative example 2 and Roche's measured values.

[0062] Figure 7 It is a comparison chart of comparative example 3 and Roche's measured values.

[0063] Figure 8 It is a comparison chart of comparative example 4 and Roche's measured values.

[0064] Figure 9 It is a comparison chart of comparative example 5 and Roche's measured values.

[0065] Figures 1-9 The horizontal axis represents the test results of the Roche estradiol detection kit (unit: pg / mL), and the vertical axis represents the test results of the immunoassay kit containing different dissociation agents in this example (unit: pg / mL). DETAILED DESCRIPTION

[0066] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of protection claimed in this application. Those skilled in the art may make various changes and modifications to the invention of this application based on the disclosed content, and such changes and modifications should also fall within the scope of protection claimed in this application.

[0067] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. The contents described below are all by mass.

[0068] The manufacturer and product number of the raw material components

[0069] Dihydrotestosterone was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. with the product number ZC-55847;

[0070] Danazol was purchased from Alfa, product number R10037;

[0071] Mesterolone was purchased from Aladdin, product number M413080-25 mg;

[0072] Perfluorooctanoic acid was purchased from Sigma, product number 171468-25g;

[0073] Levonorgestrel was purchased from Sigma-Aldrich with the product number L0551000;

[0074] Magnetic particles were purchased from Agilent, catalog number PL6727-1005;

[0075] Biotin was purchased from Thermo Fisher, catalog number 21312;

[0076] Acridinium ester was purchased from Maxtor Wai Company with the product number MTW-CLIA-007;

[0077] The Roche estradiol detection kit was purchased from Roche Diagnostics (Shanghai) Co., Ltd. with the catalog number 06656021190.

[0078] Testing instruments:

[0079] Roche cobasE411 electrochemiluminescence fully automatic immunoassay analyzer;

[0080] VIRTUOSO CS-1500 instrument.

[0081] Antibody preparation: Dilute an appropriate amount of estradiol antibody with 0.1M phosphate buffer (pH 8.0) to a final concentration of 5 mg / mL. Add biotin to a final concentration of 2 mg / mL. Mix thoroughly and incubate at room temperature for 1 hour. Purify by ultrafiltration and centrifugation to obtain biotin-labeled estradiol antibody.

[0082] Antigen preparation method: Take 0.2 mg of E2 antigen and adjust the concentration to 2 mg / mL using 0.01 M pH 8.2 phosphate buffer (PBs). Add acridinium ester at a molar ratio of antigen: acridinium ester = 1:13, react at room temperature for 1 hour, ultrafilter six times through 0.01 M pH 7.32 phosphate buffer (PBs), add an equal amount of glycerol, and store below -15°C.

[0083] Preparation method of chemiluminescent substrate:

[0084] The chemiluminescent substrate includes buffer 1 and buffer 2, wherein the buffer 1 includes 0.01M HNO3 and 0.45% H2O2; and the buffer 2 includes 0.45M NaOH.

[0085] Example 1

[0086] The dissociating agents are as follows:

[0087] 0.01 mg / L perfluorooctanoic acid, 60 mM Tris-HCl buffer, pH 8.0.

[0088] Prepare detection kit 1:

[0089] Dissociation agent 20 μL, estradiol magnetic separation reagent 20 L, estradiol antibody reagent 20 mL, estradiol antigen reagent 20 mL, chemiluminescent substrate reagent 100 mL.

[0090] Example 2

[0091] The difference between Example 2 and Example 1 is that the concentration of perfluorooctanoic acid in the dissociation agent is 0.05 mg / L, and the rest are the same.

[0092] Example 3

[0093] The difference between Example 3 and Example 1 is that the concentration of perfluorooctanoic acid in the dissociation agent is 0.5 mg / L, and the rest are the same.

[0094] Example 4

[0095] The difference between Example 4 and Example 1 is that the concentration of perfluorooctanoic acid in Example 4 is 1.0 mg / L, and the rest are the same.

[0096] Comparative Example 1

[0097] The difference between Comparative Example 1 and Example 2 is that the components and concentrations of the dissociating agent are different, and the rest are the same.

[0098] The dissociating agents are as follows:

[0099] Contains 100ug / L danazol, 2mg / L levonorgestrel, 0.05mol MES buffer, pH 6.0.

[0100] Comparative Example 2

[0101] The difference between Comparative Example 2 and Example 2 is that the components and concentrations of the dissociating agent are different, and the rest are the same.

[0102] The dissociating agents are as follows:

[0103] 200ug / L dihydrotestosterone, 2mg / L levonorgestrel, 0.1mol PB buffer, pH 6.5.

[0104] Comparative Example 3

[0105] The difference between Comparative Example 3 and Example 2 is that the components and concentrations of the dissociating agent are different, and the rest are the same.

[0106] The dissociating agents are as follows:

[0107] 50ug / mL danazol, 0.1mg / mL mesterolone, 50mM PBS buffer, pH 7.4.

[0108] Comparative Example 4

[0109] The difference between Comparative Example 4 and Example 2 is that the components and concentrations of the dissociating agent are different, and the rest are the same.

[0110] The composition of the dissociation agent without dissociation components is as follows: the buffer used to prepare the releasing agent, specifically 50 mM PBS buffer, pH 7.4.

[0111] Comparative Example 5

[0112] The difference between Comparative Example 5 and Example 2 is that the concentration of perfluorooctanoic acid in Comparative Example 5 is 2 mg / L, and the rest are the same.

[0113] Sample testing method:

[0114] A total of 150 clinical serum samples from both men and women were collected, with uniform concentrations. The samples were assigned values ​​using a Roche estradiol assay kit (electrochemiluminescence). The samples were then tested using immunoassay kits containing the nine different sample dissociation agents described above, and correlation curves were plotted.

[0115] Instructions for using the kit:

[0116] First, prepare 200 reagents of each of the nine estradiol immunoassays described above. These reagents were loaded into the reagent compartment of the VIRTUOSO CS-1500 instrument and tested on 150 clinical serum samples using each of the nine estradiol immunoassays. Estradiol concentrations in these 150 clinical serum samples were also measured using the Roche Estradiol Assay Kit (electrochemiluminescence) on a Roche cobas E411 electrochemiluminescence automated immunoassay analyzer.

[0117] Experimental results

[0118] Compare the test results of each immunoassay reagent with those of the Roche Estradiol Detection Kit (Electrochemiluminescence method), see Figures 1-9 .from Figure 8 It can be seen that the correlation coefficient R between the test results of the immunoassay kit without dissociation components and the test results of the Roche estradiol test kit is 2 Only 0.7277. Figure 2 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of Example 2 and the test results of the Roche estradiol test kit is 2 Up to 0.9845. Figure 1 It can be seen that by reducing the concentration of perfluorooctanoic acid, the correlation coefficient R between the test results of the immunoassay kit containing embodiment 1 and the test results of the Roche estradiol test kit is 2 is 0.9713. Figure 3 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of Example 3 and the test results of the Roche estradiol test kit is 2 is 0.9822. Figure 4 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of Example 4 and the test results of the Roche estradiol test kit is 2 is 0.9829. Figure 5 It can be seen that the correlation coefficient R between the test results of the immunoassay kit containing comparative example 1 and the test results of the Roche estradiol test kit is 2 is 0.9617. Figure 6 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of comparative example 2 and the test results of the Roche estradiol test kit is 2 is 0.9609. Figure 7 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of comparative example 3 and the test results of the Roche estradiol test kit is 2 is 0.9698. Figure 9 It can be seen that the correlation coefficient R between the test results of the immunoassay kit of comparative example 5 and the test results of the Roche estradiol test kit is 2 It is 0.9701.

[0119] The results demonstrate that the immunochemiluminescent quantitative detection kit prepared in Examples 1-4 for estradiol detection does not require a separate sample processing step. Instead, the test sample is simply mixed with an estradiol releaser, magnetic particles, an estradiol antibody reagent, and a magnetic estradiol antigen reagent for reaction. After the immunoreaction is complete, a chemiluminescent substrate is added, resulting in a simple procedure. Furthermore, this method achieves rapid and effective dissociation of estradiol, significantly improving the sensitivity and specificity of the estradiol immunoassay reagent.

[0120] from Figure 2 It can be seen that when only 0.05 mg / L of perfluorooctanoic acid was added in Example 2, the correlation coefficient with the Roche estradiol detection kit was higher than that of other immunoassay kits containing other release agents. The correlation coefficient R 2 It reached 0.9845, indicating that perfluorooctanoic acid has an excellent promoting effect on the release of estradiol.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of perfluorooctanoic acid in the preparation of an estradiol dissociator, characterized in that: The estradiol dissociator consists of perfluorooctanoic acid and 60 mM Tris-HCl buffer, and the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.05 mg / L.

2. An estradiol dissociating agent, characterized in that The estradiol dissociator consists of perfluorooctanoic acid and 60 mM Tris-HCl buffer; the concentration of perfluorooctanoic acid in the estradiol dissociator is 0.05 mg / L.

3. Use of the estradiol dissociating agent according to claim 2 in preparing an estradiol detection kit.

4. The use according to claim 3, characterized in that The estradiol detection kit further comprises an estradiol magnetic separation reagent, an estradiol antibody reagent, a magnetic estradiol antigen reagent and a chemiluminescent substrate reagent.

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