A kit for quantitatively detecting estradiol

Abstract

The invention discloses a kit for quantitatively detecting estradiol, which belongs to the field of biomedical testing. The kit comprises carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, a biotin-labeled E2 derivative, a dissociator, and a blocking agent. The dissociator comprises at least one of methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid, and triaton. The blocking agent comprises an anti-E1 antibody, and the dissociator comprises methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid, and triaton. The concentration of the methylhydrotestosterone, the concentration of the danazol, the concentration of the 8-aniline-1-naphthalenesulfonic acid, and the content of the triaton are 0.5% (v / v). The dissociator can effectively dissociate E2 from a state bound to a protein, and the blocking agent can effectively combine with interfering substances in a sample to effectively eliminate interference with the detection caused by substances with a structure similar to E2, thereby improving the accuracy of the detection result.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and particularly relates to a kit for quantitatively detecting estradiol. Background Art

[0002] 17β-estradiol (E2) is one of the most biologically active estrogens, with a molecular weight of 272.3 Da. In women, E2 is primarily synthesized by the ovaries, while in men, it is primarily produced by the adrenal cortex and testes. E2 is a lipid-soluble molecule. Most of the E2 secreted into the blood is specifically bound to sex hormone-binding globulin and nonspecifically to human serum albumin, with only a small fraction remaining free. Bound E2 is biologically inactive, while the small amount of free E2 can enter target cells and bind to corresponding receptors to exert its biological effects. In clinical practice, assessment of E2 levels plays an important role in the evaluation of ovarian function, the auxiliary diagnosis and treatment of ovarian hyperstimulation syndrome, and precocious puberty. It can also be used to monitor fertility treatment and determine the timing of ovulation within the framework of in vitro fertilization (IVF).

[0003] Clinical diagnosis measures the total amount of E2, not the free form. Therefore, it is necessary to release E2 bound to sex hormone-binding globulin and albumin. However, existing technologies suffer from incomplete dissociation, leading to inaccurate test results. Furthermore, the presence of interfering substances with similar structures to estradiol in the sample can affect estradiol determination, resulting in inaccurate test results. Summary of the Invention

[0004] The purpose of the present invention is to provide a kit for quantitative detection of estradiol, so as to solve the problems in the prior art of incomplete dissociation and the presence of interfering substances with similar structures to estradiol in the sample, which may affect the determination of estradiol and lead to inaccurate test results.

[0005] To achieve the above object, the present invention provides the following technical solutions: a kit for quantitatively detecting estradiol, comprising carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, a biotin-labeled E2 derivative, a dissociating agent, and a blocking agent;

[0006] The dissociating agent comprises at least one of methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid and triaton;

[0007] The blocking agent comprises an anti-E1 antibody.

[0008] Furthermore, the carboxyl magnetic beads are a mixture of magnetic beads coated with streptavidin and a buffer solution; the buffer solution is one of phosphate buffer solution containing bovine serum albumin, citrate buffer solution, Tris salt buffer solution and morpholineethanesulfonic acid buffer solution.

[0009] Furthermore, the concentration of the streptavidin-coated magnetic beads in the buffer solution was 0.25 mg / mL.

[0010] Furthermore, the dissociation agent uses methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid and triaton, the concentration of methylhydrotestosterone is 0.1 μg / mL, the concentration of danazol is 0.1 μg / mL, the concentration of 8-aniline-1-naphthalenesulfonic acid is 0.1 μg / mL, and the content of triaton is 0.5% (v / v).

[0011] Furthermore, the concentration of the anti-E1 antibody is 0.2 μg / mL.

[0012] Furthermore, the acridinium ester-labeled anti-E2 antibody is a mixture of an acridinium ester-anti-E2 antibody complex and a buffer solution, and the concentration of the acridinium ester-anti-E2 antibody complex in the buffer solution is 0.5±0.2 μg / mL.

[0013] Furthermore, the biotin-labeled E2 derivative is a mixture of a biotin-E2-BSA-Bio complex and a buffer solution, and the concentration of the biotin-E2-BSA-Bio complex in the buffer solution is 0.5±0.2 μg / mL.

[0014] Furthermore, the preparation of the acridinium ester-labeled E2 antibody comprises the following steps:

[0015] Take a centrifuge tube and add the anti-E2 antibody to be labeled, add the acridinium ester solution to the wall of the tube, measure the labeling buffer to flush it, blow and shake, seal it, react at 37℃ for 60 minutes, add the stop buffer after centrifugation, and react for another 30 minutes in the same operation. Transfer the product to a dialysis bag, dialyze the solution at 4℃ until the luminescence value reaches the standard, measure the protein concentration after recovery, add protein protection solution and propylene glycol to mix.

[0016] Furthermore, the preparation of the biotin-labeled E2 derivative comprises the following steps:

[0017] Take a centrifuge tube and add E2 derivative, add biotin solution to the tube wall, then flush with labeling buffer, blow and shake, then seal, react at 37.0℃ for 30 minutes, centrifuge and add stop buffer, repeat the operation and react for another 30 minutes, transfer the product to a dialysis bag, dialyze at 4℃ and change the liquid 5 times; after recovery, measure the protein concentration, add protein protection liquid and propylene glycol to mix.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The kit for quantitatively detecting estradiol can effectively dissociate E2 from the state bound to the protein through a dissociating agent, and can effectively eliminate the interference of substances with similar structures to E2 on the detection by binding with interfering substances in the sample through a blocking agent, thereby improving the accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart between Experiment 1 and Roche's measurement.

[0021] Figure 2 This is a comparison chart between Experiment 2 and Roche's measurement.

[0022] Figure 3 This is a comparison chart between Experiment 3 and Roche's measurement.

[0023] Figure 4 This is a comparison chart between Experiment 4 and Roche's measurement.

[0024] Figure 5 This is a comparison chart of the low-value sample in Experiment 4 (concentration <200pg / mL) and the Roche test value. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the embodiments.

[0026] The following examples are intended to illustrate the present invention but are not intended to limit the scope of protection of the present invention. The conditions in the examples may be further adjusted according to specific conditions. Simple improvements to the method of the present invention within the scope of the present invention are also within the scope of protection claimed in the present invention.

[0027] The present invention provides a kit for quantitatively detecting estradiol, comprising carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, and a biotin-labeled E2 derivative, as well as a dissociating agent and a blocking agent; the dissociating agent is used to dissociate E2 from a state bound to a protein; and the blocking agent is used to bind to substances in a sample that have a similar structure to E2, thereby eliminating the influence of these interfering substances on the detection results.

[0028] Carboxyl magnetic beads: Carboxyl magnetic beads are a mixture of magnetic beads coated with streptavidin and a buffer solution; the buffer solution is one of phosphate buffer, citrate buffer, Tris salt buffer, and morpholineethanesulfonic acid buffer containing bovine serum albumin. The concentration of the streptavidin-coated magnetic beads in the buffer solution is 0.25 mg / mL.

[0029] Acridinium ester-labeled anti-E2 antibody: The acridinium ester-labeled anti-E2 antibody is a mixture of an acridinium ester-anti-E2 antibody complex and a buffer solution, wherein the concentration of the acridinium ester-anti-E2 antibody complex in the buffer solution is 0.5±0.2 μg / mL.

[0030] Biotin-labeled E2 derivative: The biotin-labeled E2 derivative is a mixture of a biotin-E2-BSA-Bio complex and a buffer solution. The concentration of the biotin-E2-BSA-Bio complex in the buffer solution is 0.5±0.2 μg / mL.

[0031] Preparation of acridinium ester-labeled E2 antibody:

[0032] To a 2 mL centrifuge tube, add the anti-E2 antibody to be labeled. Accurately measure the acridinium ester solution and add it to the inner wall of the middle part of the 2 mL centrifuge tube, allowing the acridinium ester solution to hang on the tube wall and not mix with the previously added antibody. Accurately measure the labeling buffer and flush the acridinium ester solution from the tube wall to the bottom of the centrifuge tube. Repeatedly pipette up and down 3-5 times, gently shake on a vortex mixer, seal the centrifuge tube with parafilm, and place the centrifuge tube on a rotating incubator in a constant temperature incubator at 37.0°C for 60 minutes. After the reaction is complete, gently centrifuge in a centrifuge. Accurately measure the stop buffer and add it to the centrifuge tube. Repeatedly pipette up and down 3-5 times, gently shake on a vortex mixer, seal the centrifuge tube with parafilm, and place the centrifuge tube on a rotating incubator in a constant temperature incubator at 37.0°C for 30 minutes. Transfer the labeled antibody that has reacted well into a dialysis bag and dialyze it with phosphate buffer at 4°C in a 2L beaker. Replace the dialysis buffer every 4 hours until the luminescence value reaches the standard and the dialysis is completed. After the dialysis is completed, recover the antibody from the dialysis bag and record the recovered volume. After determining the protein concentration, add 1 / 3 volume of protein protection solution and mix well. Finally, add an equal volume (antibody + protein protection solution) of propylene glycol and mix well. After preparation, write down the name, batch number, quantity, concentration, etc. and set aside.

[0033] Preparation of biotinylated E2 derivatives:

[0034] Take a 2mL centrifuge tube and add the E2 derivative to be labeled. Accurately measure the biotin solution and add it to the inner wall of the middle part of the 2mL centrifuge tube, allowing the biotin solution to hang on the tube wall. Do not mix with the previously added E2 derivative. Accurately measure the labeling buffer and flush the biotin solution on the tube wall to the bottom of the centrifuge tube along the tube wall. Use a pipette to repeatedly pipette 3-5 times, gently shake on a vortex mixer, seal the centrifuge tube with sealing film, and then place the centrifuge tube on a rotating incubator in a constant temperature incubator and react at 37.0℃ for 30 minutes. After the reaction is completed, gently centrifuge in a centrifuge, accurately measure the stop buffer and add it to the centrifuge tube. Use a pipette to repeatedly pipette 3-5 times, gently shake on a vortex mixer, seal the centrifuge tube with sealing film, and then place the centrifuge tube on a rotating incubator in a constant temperature incubator and react at 37.0℃ for 30 minutes. Transfer the reacted labeled derivative to a dialysis bag and dialyze it in a 2L beaker at 4°C against phosphate buffer. Change the dialysis buffer every 4 hours for 5 changes. After dialysis, recover the E2 derivative from the dialysis bag and record the recovered volume. After determining the protein concentration, add 1 / 3 volume of protein protection solution and mix thoroughly. Finally, add an equal volume of glycerol (E2 derivative + protein protection solution) and mix thoroughly. After preparation, indicate the name, batch number, quantity, concentration, etc. and set aside.

[0035] Preparation of dissociation and blocking agents:

[0036] Example 1: Preparation of a solution containing an estradiol dissociating agent

[0037] Weigh 19.524 g of morpholineethanesulfonic acid (MES) and dissolve it in 1 L of deionized water. Add the components according to Table 1, followed by 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5 mol / L NaOH, and store the solution at 4°C until ready for use. This prepared dissociation agent is designated Group ①.

[0038] Table 1 Estradiol dissociating agents

[0039] Chemical name concentration Methylhydrotestosterone 0.1 μg / mL Danazol 0.1 μg / mL 8-Anilino-1-naphthalenesulfonic acid 0.1 μg / mL Triton 0.5% (v / v)

[0040] Example 2: Preparation of a solution containing an estradiol blocker

[0041] Weigh 19.524g of morpholineethanesulfonic acid (MES) and dissolve it in 1L of deionized water. Add the components according to Table 2, followed by 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and store the solution at 4°C until ready to use. This blocker is designated Group ②.

[0042] Table 2 Estradiol blockers

[0043] Chemical name concentration Anti-E1 antibody 0.1 μg / mL

[0044] Example 3: Preparation of a solution containing both an estradiol dissociating agent and a blocking agent

[0045] Weigh 19.524 g of morpholineethanesulfonic acid (MES) and dissolve it in 1 L of deionized water. Add the components according to Table 3, followed by 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5 mol / L NaOH, and store the solution at 4°C until ready to use. This dissociator and blocker combination is designated Group ③.

[0046] Table 3 Estradiol dissociators + blockers

[0047]

[0048]

[0049] Example 4: Preparation of a solution containing mesterolone, testosterone, and danazol as dissociating agents and an estrone antibody as a blocking agent

[0050] Weigh 19.524 g of morpholineethanesulfonic acid (MES) and dissolve it in 1 L of deionized water. Add the components according to Table 4, followed by 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5 mol / L NaOH, and store the solution at 4°C until ready to use. This dissociator and blocker combination is designated Group ④.

[0051] Table 4 Estradiol dissociators + blockers

[0052] Chemical name concentration Mesterolone 0.05 μg / mL testosterone 0.05 μg / mL Danazol 0.05 μg / mL Estrone antibodies 0.1 μg / mL

[0053] Weigh 19.524 g of morpholineethanesulfonic acid (MES) and dissolve it in 1 L of deionized water. Add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5 mol / L NaOH and store the solution at 4°C until ready for use. This buffer solution is designated Group 5 and serves as a blank control.

[0054] See also Figure 1-5 ,The following experiments were conducted based on Examples 1-4.

[0055] Experiment 1

[0056] The accuracy of the kit was compared between the solution containing estradiol dissociator (Group ①) and the blank control (Group ⑤) under the same conditions.

[0057] Experimental materials: carboxyl magnetic beads (magnetic beads purchased from Hangzhou Boyue), acridinium ester-labeled anti-E2 antibodies, biotin-labeled E2 derivatives, Yilanbei fully automatic instrument excitation solution, Yilanbei cleaning solution, Yilanbei fully automatic chemiluminescence immunoassay, methylhydrotestosterone, danazol, 8-anilino-1-naphthalenesulfonic acid, Triton, anti-E1 antibodies, 40 fresh E2 clinical samples and a third-party clinical test report (Roche).

[0058] The method of Example 1 was used to prepare a kit comprising a solution containing a dissociating agent, carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, and a biotin-labeled E2 derivative. Samples S1-S40 were measured and compared with the Roche value.

[0059] The experimental results are shown in Tables 1 and 2.

[0060] Table 1 Results of Roche samples

[0061]

[0062]

[0063] Table 2 Correlation with Roche values

[0064]

[0065]

[0066] Experimental conclusion: After adding the dissociation agent, the correlation increased compared with the Roche value, but there were still some interfering samples, resulting in a large deviation between the test results and the Roche value.

[0067] Experiment 2

[0068] The accuracy of the kit was compared between the solution containing estradiol blocker (Group ②) and the blank control (Group ⑤) under the same conditions.

[0069] Using the method of Example 2, a solution containing a blocking agent was combined with carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, and a biotin-labeled E2 derivative to form a kit. Samples S1-S40 were measured and compared with the Roche value.

[0070] The experimental results are shown in Tables 3 and 4.

[0071] Table 3 Results of Roche samples

[0072]

[0073]

[0074]

[0075] Table 4 Correlation with Roche values

[0076] Grouping Linear equations Correlation coefficient r Group ② y=0.9593x+12.329 0.9929 ⑤ Group y=0.9916x+30.716 0.9722

[0077] Experimental conclusion: After adding the blocker, the test results of the samples with larger test deviation in group ⑤ were compared with the Roche test values, and the deviation was significantly reduced.

[0078] Experiment 3

[0079] The accuracy of the kit was compared between the solution containing estradiol dissociator and blocker (Group ③) and the blank control (Group ⑤) under the same conditions.

[0080] Using the method of Example 3, a solution containing a dissociating agent and a blocking agent was combined with carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, and a biotin-labeled E2 derivative to form a kit. Samples S1-S40 were measured and compared with the Roche value.

[0081] The experimental results are shown in Tables 5 and 6.

[0082] Table 5 Results of Roche samples

[0083]

[0084]

[0085]

[0086] Table 6 Correlation with Roche values

[0087] Grouping Linear equations Correlation coefficient r Group ③ y=0.9864x+4.0818 0.9993 ⑤ Group y=0.9916x+30.716 0.9722

[0088] Experimental conclusion: After adding dissociation agent and blocking agent at the same time, the correlation increased significantly, the deviation between sample results and Roche values ​​decreased, and the accuracy of serum testing by the kit was improved.

[0089] The above three experiments show that after adding dissociation agents and blocking agents during the use of the kit, when testing clinical samples, the correlation is significantly improved, the deviation is reduced, and the accuracy is improved compared with the Roche value.

[0090] Comparative Experiment 4

[0091] A comparison experiment of the kit accuracy was conducted under the same conditions between a solution containing estradiol dissociators (methylhydrotestosterone, danazol, 8-anilino-1-naphthalenesulfonic acid and triaton) and a blocker (anti-E1 antibody) (Group ③) and a solution containing estradiol dissociators (methyltestosterone, testosterone and danazol) and a blocker (estrone antibody) (Group ④).

[0092] The methods of Example 3 and Example 4 were used to prepare a kit comprising a solution containing a dissociating agent and a blocking agent, carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, and a biotin-labeled E2 derivative. Sample S1-S40 was measured and compared with the Roche value.

[0093] The experimental results are shown in Tables 7 and 8.

[0094] Table 7 Results of Roche samples

[0095]

[0096]

[0097] Table 8 Correlation with Roche values

[0098] Grouping Linear equations Correlation coefficient r Group ③ y=0.9864x+4.0818 0.9993 Group ④ y=0.9483x+13.017 0.9942

[0099] Experimental conclusion: The solution containing estradiol dissociation agents (methylhydrotestosterone, danazol, 8-anilino-1-naphthalenesulfonic acid and triaton) and blocking agent (anti-E1 antibody) (Group ③) and the solution containing estradiol dissociation agents (methyltestosterone, testosterone and danazol) and blocking agent (estrone antibody) (Group ④) were tested on Roche serum under the same conditions. The results show that the correlation of Group ③ is better, and the deviation of the low value part with the Roche serum concentration is smaller. Figure 5 It can be seen that the low value (concentration < 200 pg / mL) data of group ④ is more discrete than the data of group ③.

[0100] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. A person skilled in the art may make other changes or modifications based on the above description, and these obvious changes or modifications derived from the spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A kit for quantitatively detecting estradiol, characterized in that: The method comprises carboxyl magnetic beads, an acridinium ester-labeled anti-E2 antibody, a biotin-labeled E2 derivative, a dissociation agent, and a blocking agent; The blocking agent comprises an anti-E1 antibody; The carboxyl magnetic beads are a mixture of magnetic beads coated with streptavidin and a buffer solution; the buffer solution is one of phosphate buffer, citrate buffer, Tris salt buffer and morpholineethanesulfonic acid buffer containing bovine serum albumin; The dissociation agent comprises methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid and triaton, wherein the concentration of methylhydrotestosterone is 0.1 μg / mL, the concentration of danazol is 0.1 μg / mL, the concentration of 8-aniline-1-naphthalenesulfonic acid is 0.1 μg / mL, and the content of triaton is 0.5% (v / v).

2. The kit for quantitatively detecting estradiol according to claim 1, wherein: The concentration of streptavidin-coated magnetic beads in the buffer was 0.25 mg / mL.

3. The kit for quantitatively detecting estradiol according to claim 1, wherein: The concentration of the anti-E1 antibody was 0.2 μg / mL.

4. The kit for quantitatively detecting estradiol according to claim 1, wherein: The acridinium ester-labeled anti-E2 antibody is a mixture of an acridinium ester-anti-E2 antibody complex and a buffer solution, and the concentration of the acridinium ester-anti-E2 antibody complex in the buffer solution is 0.5±0.2 μg / mL.

5. The assay kit for quantitatively detecting estradiol according to claim 1, wherein: The biotin-labeled E2 derivative is a mixture of a biotin-E2-BSA-Bio complex and a buffer solution, and the concentration of the biotin-E2-BSA-Bio complex in the buffer solution is 0.5±0.2 μg / mL.

6. The assay kit for quantitatively detecting estradiol according to claim 1, wherein: The preparation of the acridinium ester-labeled E2 antibody comprises the following steps: Take a centrifuge tube and add the anti-E2 antibody to be labeled, add the acridinium ester solution to the wall of the tube, measure the labeling buffer to flush it down, blow and shake, seal it, react at 37℃ for 60 minutes, add the stop buffer after centrifugation, and react for another 30 minutes in the same operation. Transfer the product to a dialysis bag, dialyze the solution at 4℃ until the luminescence value reaches the standard, measure the protein concentration after recovery, add protein protection solution and propylene glycol to mix.

7. The assay kit for quantitatively detecting estradiol according to claim 1, wherein: The preparation of the biotin-labeled E2 derivative comprises the following steps: Take a centrifuge tube and add E2 derivative, add biotin solution to the tube wall, then flush with labeling buffer, blow and shake, then seal, react at 37.0℃ for 30 minutes, centrifuge and add stop buffer, repeat the operation and react for another 30 minutes, transfer the product to a dialysis bag, dialyze at 4℃ and change the solution 5 times; after recovery, measure the protein concentration, add protein protection solution and propylene glycol to mix.

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