Determination kit for quantitative detection of estradiol

Abstract

Belonging to the field of biomedical detection, the invention discloses a determination kit for quantitative detection of estradiol, which comprises carboxyl magnetic beads, an acridinium ester labeled anti-E2 antibody, a biotin labeled E2 derivative, a dissociation agent and a blocking agent. The dissociation agent is prepared from at least one of methylhydrotestosterone, danazol, 8-aniline-1-naphthalene sulfonic acid and triton; the blocking agent comprises an anti-E1 antibody, the dissociation agent adopts methyltestosterone, danazol, 8-aniline-1-naphthalene sulfonic acid and triton, the concentration of methyltestosterone is 0.1 mu g / mL, the concentration of danazol is 0.1 mu g / mL, the concentration of 8-aniline-1-naphthalene sulfonic acid is 0.1 mu g / mL, and the content of triton is 0.5% (v / v); according to the present invention, the E2 can be well dissociated from the protein binding state through the dissociation agent, and the dissociation agent is combined with the interfering substance in the sample so as to well eliminate the interference of the substance similar to the E2 structure on the detection, such that the accuracy of the detection result is improved;

Description

Technical Field

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

[0002] 17β-estradiol (E2) is one of the most biologically active estrogens with a molecular weight of 272.3Da. In women, E2 is mainly synthesized by the ovaries, while in men, it is mainly produced by the adrenal cortex and testes. E2 is a fat-soluble molecule. Most of the E2 secreted into the blood is specifically bound to sex hormone-binding globulin and nonspecifically bound to human serum albumin, and only a small part is in a free state. The bound E2 has no biological activity, and a small amount of E2 in a free state can enter the target cells and bind to the corresponding receptors to exert its biological effects. In clinical work, the assessment of E2 levels plays an important role in the evaluation of ovarian function, ovarian hyperstimulation syndrome, and the auxiliary diagnosis and treatment of precocious puberty. It can also monitor fertility treatment and determine the time of ovulation within the framework of in vitro fertilization (IVF).

[0003] In clinical diagnosis, the total amount of E2 is measured, not the free E2, so the E2 bound to sex hormone binding globulin and albumin needs to be released, but the existing technology has the problem of incomplete dissociation, resulting in inaccurate test results. At the same time, the presence of interfering substances with similar structures to estradiol in the sample will affect the determination of estradiol, resulting in inaccurate test results. Summary of the invention

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

[0005] To achieve the above object, the present invention provides the following technical solutions: a kit for quantitative detection of estradiol, comprising carboxyl magnetic beads, an anti-E2 antibody labeled with an acridinium ester, an E2 derivative labeled with biotin, 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; the buffer is one of phosphate buffer, citrate buffer, Tris salt buffer and morpholineethanesulfonic acid buffer containing bovine serum albumin.

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

[0010] Furthermore, the dissociating agent is methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid and Triton, 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 Triton 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 and 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 at 4℃ until the luminescence value reaches the standard, measure the protein concentration after recovery, and add protein protection solution and propylene glycol to mix well.

[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 rinse with labeling buffer, blow and shake, then seal, react at 37.0℃ for 30 minutes, centrifuge and add stop buffer, do the same operation and react for another 30 minutes, transfer the product to a dialysis bag, dialyze at 4℃ and change the solution 5 times; measure the protein concentration after recovery, add protein protection solution and propylene glycol to mix well.

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

[0019] The kit for quantitatively detecting estradiol can well dissociate E2 from the state of being bound to the protein through a dissociating agent, and can well eliminate the interference of substances with similar structures to E2 on the detection through a blocking agent by binding to interfering substances in the sample, thereby improving the accuracy of the detection result. 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 values.

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

[0025] The present invention will be further described below in conjunction with the embodiments.

[0026] The following examples are used to illustrate the present invention, but they cannot be used to limit the scope of protection of the present invention. The conditions in the examples can be further adjusted according to specific conditions. Simple improvements to the method of the present invention under the premise of the concept of the present invention belong to the scope of protection claimed in the present invention.

[0027] The invention provides a kit for quantitatively detecting estradiol, comprising carboxyl magnetic beads, an anti-E2 antibody labeled with an acridinium ester and an E2 derivative labeled with biotin, and also comprising a dissociating agent and a blocking agent; the dissociating agent is used for dissociating E2 from a state of being bound to a protein; and the blocking agent is used for binding substances in a sample that are structurally similar to E2, thereby eliminating the influence of these interfering substances on the detection result.

[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, and the concentration of the magnetic beads coated with streptavidin 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, wherein 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] Take a 2mL centrifuge tube, add the anti-E2 antibody to be labeled, accurately measure the acridinium ester solution, and then add it to the inner wall of the middle part of the 2mL centrifuge tube, so that the acridinium ester solution hangs on the tube wall, and does not mix with the antibody added first, accurately measure the labeling buffer, flush the acridinium ester solution on the tube wall to the bottom of the centrifuge tube along the tube wall of the centrifuge tube, repeatedly blow 3-5 times with a pipette, gently shake on a vortex mixer, seal the centrifuge tube with a sealing film, and then place the centrifuge tube on a rotating incubator placed in a constant temperature incubator, and react at 37.0℃ for 60 minutes. After the reaction is completed, centrifuge gently in a centrifuge, accurately measure the stop buffer, add it to the centrifuge tube, repeatedly blow 3-5 times with a pipette, gently shake on a vortex mixer, seal the centrifuge tube with a sealing film, and then place the centrifuge tube on a rotating incubator placed in a constant temperature incubator, and react at 37.0℃ 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. for use.

[0033] Preparation of biotinylated E2 derivatives:

[0034] Take a 2mL centrifuge tube, add the E2 derivative to be labeled, accurately measure the biotin solution, and then add it to the inner wall of the middle part of the 2mL centrifuge tube, so that the biotin solution hangs on the tube wall, and does not mix with the E2 derivative added first, 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 of the centrifuge tube, repeatedly blow 3-5 times with a pipette, gently shake on a vortex mixer, seal the centrifuge tube with a sealing film, and then place the centrifuge tube on a rotating incubator placed in a constant temperature incubator, and react at 37.0℃ for 30 minutes. After the reaction is completed, centrifuge gently in a centrifuge, accurately measure the stop buffer, add it to the centrifuge tube, repeatedly blow 3-5 times with a pipette, gently shake on a vortex mixer, seal the centrifuge tube with a sealing film, and then place the centrifuge tube on a rotating incubator placed in a constant temperature incubator, and react at 37.0℃ for 30 minutes. Transfer the labeled derivative 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. Replace it 5 times and the dialysis is over. After the dialysis is over, 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 well. Finally, add an equal volume of propylene glycol (E2 derivative + protein protection solution) and mix well. After preparation, write down the name, batch number, quantity, concentration, etc. for use.

[0035] Preparation of dissociation and blocking agents:

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

[0037] Weigh 19.524g of morpholineethanesulfonic acid (MES), dissolve it in 1L of deionized water, add the components according to Table 1, and then add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and place the solution at 4℃ for later use. The above prepared dissociation agents are numbered as group ①.

[0038] Table 1 Estradiol dissociating agents

[0039] Chemical name concentration Methylhydrotestosterone 0.1 μg / mL Danazol 0.1 μg / mL 8-Aniline-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), dissolve in 1L of deionized water, add the components according to Table 2, and then add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and place the solution at 4℃ for later use. The blockers prepared above are numbered as 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 19.524g of morpholineethanesulfonic acid (MES), dissolve it in 1L of deionized water, add the components according to Table 3, and then add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and place the solution at 4℃ for later use. The above-prepared dissociation agent and blocking agent combination is numbered as group ③.

[0046] Table 3 Estradiol dissociators + blockers

[0047]

[0048]

[0049] Example 4: Preparation of a solution in which the dissociating agents are mesterolone, testosterone, and danazol and the blocking agent is an estrone antibody

[0050] Weigh 19.524g of morpholineethanesulfonic acid (MES), dissolve in 1L of deionized water, add the components according to Table 4, and then add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and place the solution at 4℃ for later use. The above-prepared dissociation agent and blocking agent combination is numbered as 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 Antibody 0.1 μg / mL

[0053] Weigh 19.524g of morpholineethanesulfonic acid (MES), dissolve in 1L of deionized water, and then add 0.1% (v / v) Proclin 300. Adjust the pH to 5.50 with 5mol / L NaOH, and place the solution at 4℃ for later use. The buffer prepared above is numbered as group ⑤ and used 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-aniline-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 adopted to form a kit with a solution containing a dissociating agent, carboxyl magnetic beads, acridinium ester-labeled anti-E2 antibodies, and biotin-labeled E2 derivatives, and the 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 determination 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] The method of Example 2 was adopted to form a kit by combining a solution containing a blocking agent with carboxyl magnetic beads, an anti-E2 antibody labeled with an acridinium ester, and an E2 derivative labeled with biotin, and the 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] The method of Example 3 was adopted to form a kit with 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, and the 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] Through the above three experiments, it can be concluded that after adding dissociation agents and blocking agents during the use of the kit, when testing clinical samples, compared with the Roche value, the correlation is significantly improved, the deviation is reduced, and the accuracy is improved.

[0090] Comparative Experiment 4

[0091] The accuracy of the kit was compared 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 respectively used to form a kit with 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. Samples S1-S40 were 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 dissociators (methylhydrotestosterone, danazol, 8-anilino-1-naphthalenesulfonic acid and triaton) and blocker (anti-E1 antibody) (Group ③) and the solution containing estradiol dissociators (methyltestosterone, testosterone and danazol) and blocker (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 from the Roche serum concentration is smaller. Figure 5 It can be seen that the low value (concentration <200pg / mL) of group ④ data is more discrete than that of group ③ data.

[0100] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the embodiments of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made 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: It includes carboxyl magnetic beads, acridinium ester labeled anti-E2 antibody, biotin labeled E2 derivative, dissociation agent and blocking agent; The dissociating agent comprises at least one of methylhydrotestosterone, danazol, 8-aniline-1-naphthalenesulfonic acid and triaton; The blocking agent comprises an anti-E1 antibody.

2. The kit for quantitatively detecting estradiol according to claim 1, characterized in that: The carboxyl magnetic beads are a mixture of magnetic beads coated with streptavidin and a buffer solution; the buffer solution is one of a phosphate buffer solution containing bovine serum albumin, a citrate buffer solution, a Tris salt buffer solution and a morpholineethanesulfonic acid buffer solution.

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

4. The kit for quantitatively detecting estradiol according to claim 1, characterized in that: The dissociation agent is 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).

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

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

7. The kit for quantitatively detecting estradiol according to claim 1, characterized in that: 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.

8. The kit for quantitatively detecting estradiol according to claim 1, characterized in that: 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 and 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 at 4℃ until the luminescence value reaches the standard, measure the protein concentration after recovery, and add protein protection solution and propylene glycol to mix well.

9. The kit for quantitatively detecting estradiol according to claim 1, characterized in that: 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 rinse with labeling buffer, blow and shake, then seal, react at 37.0℃ for 30 minutes, centrifuge and add stop buffer, do the same operation and react for another 30 minutes, transfer the product to a dialysis bag, dialyze at 4℃ and change the solution 5 times; measure the protein concentration after recovery, add protein protection solution and propylene glycol to mix well.

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