Test strip for combined detection of AMH and FSH and preparation method thereof
By preparing test strips that jointly detect AMH and FSH, the problems of cumbersome and high cost of existing detection methods are solved, and the operation and cost reduction are achieved. The AMH and FSH can be detected simultaneously, and the needs of ovarian reserve function assessment and female reproductive health monitoring are met.
Patent Information
- Application Number
- CN202510588399.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-08-15
AI Technical Summary
The existing methods for detecting AMH and FSH are cumbersome, costly, and inaccurate, which cannot meet clinical needs. Single indicator detection cannot meet the comprehensive testing needs of ovarian reserve function evaluation.
Test strips that jointly detect AMH and FSH, including pretreated binding pads, sample pads and nitrocellulose membranes, were used to detect using fluorescent microsphere-labeled antibodies, combined with automatic cutting mechanisms, simplify operation and reduce costs.
It has achieved simplified operations and reduced costs, and can detect AMH and FSH indicators simultaneously, reduce multiple detection errors. It is suitable for clinical diagnosis and home self-tests, and comprehensively evaluates women's fertility.
Smart Images
Figure CN120490467A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of in vitro immunodiagnosis, and particularly relates to a test strip for jointly detecting AMH and FSH and a preparation method thereof. Background Art
[0002] Anti-Müllerian hormone (AMH) is a glycoprotein hormone secreted by gonadal Sertoli cells. It belongs to the transforming growth factor β superfamily and is of great significance in the development of the reproductive system. In women, it is secreted by ovarian granulosa cells and is closely related to ovarian reserve function. It can inhibit the recruitment of primordial follicles and the development of antral follicles, preventing premature follicle exhaustion. In men, AMH is secreted by testicular Sertoli cells, inducing Müllerian duct degeneration during the embryonic period. After puberty, levels gradually decrease, reflecting the function of testicular Sertoli cells. Clinically, different conditions correspond to different AMH cutoff levels. For example, when the ovarian reserve function of women over 40 years old is reduced, the AMH cutoff value is 0.5-1.1μg / L. The Poseidon standard uses AMH <1.2μg / L as a predictor of poor ovarian response. The serum AMH level of patients with polycystic ovary syndrome is 2-3 times higher than that of normal women of the same age.
[0003] Follicle-stimulating hormone (FSH) is a glycoprotein hormone secreted by basophils in the anterior pituitary gland and plays a key role in the human reproductive system. In women, it promotes follicular development and maturation, synergizing with LH to stimulate estrogen secretion and ovulation, and participates in normal menstruation. In men, it promotes seminiferous duct formation and spermatogenesis. FSH secretion is controlled by hypothalamic gonadotropin-releasing hormone and is also regulated by ovarian estrogen. Its blood concentration and urinary excretion fluctuate during the menstrual cycle, with an increase in excretion after menopause. Blood FSH concentrations range from 1.5 to 10 mIU / mL in the preovulatory phase, 8 to 20 mIU / mL during the ovulatory phase, and 2 to 10 mIU / mL in the postovulatory phase. Normal values are generally between 5 and 40 mIU / mL.
[0004] Both AMH and FSH are key indicators for assessing female ovarian function and reproductive health. AMH is widely used in the assessment of ovarian reserve function, prediction of premature ovarian failure, and auxiliary diagnosis of polycystic ovary syndrome. FSH is crucial to the reproductive function of both men and women, and detecting its levels in blood and urine can help diagnose related diseases. However, existing detection methods have defects. The enzyme-linked immunosorbent assay is cumbersome, time-consuming, and produces inaccurate results. Although the chemiluminescence method has gradually replaced the former, it is difficult to promote due to the expensive equipment, the need for professional operation and maintenance, and the high cost of testing. China relies on imported test kits, which are expensive and complicated to operate, and cannot meet clinical needs.
[0005] The immunofluorescence detection method has shown many advantages in related tests. In terms of operation, it is simple and convenient. Like a variety of test strips, you only need to add the sample to the sample well and use the immunofluorescence analyzer to read the results. No professional operation is required. The detection efficiency is high, and the results can usually be obtained in 10 to 15 minutes, which can meet the needs of rapid diagnosis. It performs well in terms of sensitivity and can detect low concentrations of target substances. For example, the detection limit of some test kits for AMH can be as low as 0.1 ng / mL, and the detection sensitivity for FSH can reach 5mIU / mL. In addition, some detection methods can detect multiple indicators at the same time, reduce the error of multiple tests, simplify operations and reduce costs. They are suitable for clinical auxiliary diagnosis and POCT testing, and are of great value in the evaluation of ovarian reserve function, diagnosis of related diseases and monitoring of female reproductive health.
[0006] However, single-indicator testing has limitations, and ovarian reserve function assessment requires a comprehensive approach. Current clinically used reference indicators or methods, such as age, hormone levels, and ultrasound examinations, cannot independently meet testing needs. Summary of the Invention
[0007] In order to solve the problems in the prior art, the present invention provides a test strip for the combined detection of AMH and FSH and a preparation method thereof, so as to achieve the combined detection of AMH and FSH indicators, with simple operation, little harm to the human body, and economical applicability, and comprehensively detect multiple biological indicators of ovarian reserve function, which can more comprehensively and accurately evaluate female fertility and meet the actual clinical needs.
[0008] The present invention solves the technical problem by adopting the following technical solutions:
[0009] The present invention aims to provide a test strip for the combined detection of AMH and FSH, comprising a conjugate pad pretreated with a conjugate pad pretreatment solution, a sample pad pretreated with a sample pad pretreatment solution, and a nitrocellulose membrane, wherein the nitrocellulose membrane is coated with an antibody diluted with a coating antibody diluent.
[0010] The conjugate pad and sample pad are both glass cellulose membranes, and the test strips also include conventional structures such as PVC sheets and absorbent pads.
[0011] Furthermore, the conjugate pad pretreatment solution includes 3% trehalose, 0.2% Tween-20, 1% BSA, 0.1% PEG20000 and 4% 50 mM borate buffer.
[0012] Furthermore, the conjugate pad pretreated with the conjugate pad pretreatment solution is also sprayed with an antibody mixture labeled with fluorescent microspheres, wherein the antibody mixture includes human AMH monoclonal antibody 1, human FSH monoclonal antibody 1, human AMH polyclonal antibody and human FSH polyclonal antibody.
[0013] Furthermore, the sample pad pretreatment liquid includes 3% trehalose, 0.2% Tween-20, 1% BSA, and 50 mM borate buffer.
[0014] Furthermore, the antibodies coated on the nitrocellulose membrane include human AMH monoclonal antibody 2 (test line 1), human FSH monoclonal antibody 2 (test line 2) and goat anti-mouse IgG monoclonal antibody (quality control line), and the coating antibody diluent includes phosphate buffered saline (0.01M PBS Ph7.2-7.4) containing 1% sodium chloride and 2% trehalose.
[0015] A method for preparing a test strip for jointly detecting AMH and FSH comprises:
[0016] Preparation of the conjugate pad: pre-treat the conjugate pad with a conjugate pad pre-treatment solution, dry it, and then spray it with a mixture of antibodies labeled with fluorescent microspheres;
[0017] Preparation of sample pad: pre-treat the sample pad with sample pad pre-treatment solution and dry it for later use;
[0018] Streak the nitrocellulose membrane: dilute the antibody with the coating antibody diluent, then streak the nitrocellulose membrane. After streaking, dry the nitrocellulose membrane for later use.
[0019] Test strip assembly: Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence, and use a fully automatic chopping machine to cut into 3.00 mm wide test strips.
[0020] Furthermore, the conjugate pad was soaked in the conjugate pad pretreatment solution for 20 minutes, and then taken out and dried in a drying oven at 40° C. for 3 hours.
[0021] Furthermore, the method of pre-treating the dried conjugate pad and spraying the mixed antibody solution labeled with fluorescent microspheres includes: centrifuging the microspheres and discarding the supernatant, then adding phosphate buffer (pH 6.0-7.0) to adjust the microsphere concentration to 0.1-1%, adding EDC and NHS with a final concentration of 0.1-5 mg / mL, activating at room temperature for 20-40 minutes, rotating at room temperature for 1.5-3 hours, centrifuging and discarding the supernatant, then dissolving with phosphate buffer (pH 6.0-7.0), ultrasonically dispersing the re-dissolved microspheres, and then adding human AMH monoclonal antibody 1, human FSH monoclonal antibody 1, and human AMH polyclonal antibody at a concentration of 0.1-1.0 mg / mL. The human AMH monoclonal antibody 1 labeling solution, the human FSH monoclonal antibody 1 labeling solution, the human AMH polyclonal antibody labeling solution and the human FSH polyclonal antibody labeling solution were obtained. The human AMH monoclonal antibody 1 labeling solution, the human FSH monoclonal antibody 1 labeling solution, the human AMH polyclonal antibody labeling solution and the human FSH polyclonal antibody labeling solution were mixed in a ratio of 1:1:(0.2-0.5):(0.2-0.5) to obtain an antibody mixture, which was then sprayed on a pretreated and dried conjugate pad at 6 μL / cm using an XYZ gold spray film instrument, and finally dried in a drying oven at 40°C for 3 h.
[0022] Furthermore, the sample pad was soaked in the sample pad pretreatment solution for 20 minutes, and then taken out and dried in a drying oven at 40° C. for 3 hours.
[0023] Furthermore, the concentration of human AMH monoclonal antibody 2 (T1) after dilution was 0.5-1 mg / mL, the concentration of human FSH monoclonal antibody 2 (T2) after dilution was 0.5-1 mg / mL, and the concentration of goat anti-mouse IgG monoclonal antibody (C) after dilution was 0.3-0.8 mg / mL. Then, an XYZ gold spray film streaker was used to streak the nitrocellulose membrane at 0.8-1 μL / cm, and the streaking lines were C, T1, and T2 respectively, and the spacing between the three lines was 0.4-0.5 mm. After streaking, the nitrocellulose membrane was placed in a drying oven at 40°C and dried for 3 hours.
[0024] Method for preparing a standard curve
[0025] (1) Preparation of standard solutions: Prepare AMH and FSH standard solutions of different concentrations.
[0026] (2) Fluorescence value reading: AMH and FSH sample solutions were tested on the reagent card respectively. After adding the samples, the card was placed in the fluorescence immunoassay analyzer. After 10 minutes, the fluorescence values of the detection lines T1, T2 and C were obtained. Each concentration was repeated three times and the average value was obtained.
[0027] (3) Establishment of standard curve: Based on the fluorescence intensity data of the test line and the quality control line, analyze the data, use the sample solution concentration as the horizontal axis and the fluorescence intensity ratio of the test line / quality control line (T1 / C or T2 / C) as the vertical axis, and fit it into a standard curve for quantitative detection of sample concentration.
[0028] How to use the test strip: Add 60 μL of blood sample to the sample addition end of the test strip, insert the test strip into the fluorescent immunoassay instrument, read the fluorescence intensity values corresponding to C, T1 and T2 after 10 minutes, and calculate the concentrations of AMH and FSH respectively according to the fluorescence intensity values and the standard curve.
[0029] Performance testing of test strips: The performance of the prepared test strips is tested, including detection limit, accuracy and specificity.
[0030] Compared with the prior art, the beneficial technical effects of the present invention are:
[0031] The reagent strip for the combined detection of AMH and FSH of the present invention can simultaneously detect AMH and FSH indicators, reduce errors in multiple detections, simplify operations and reduce costs. It is suitable for clinical diagnosis POCT testing and home self-examination, and has important value in ovarian reserve function assessment, related disease diagnosis and female reproductive health monitoring.
[0032] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 AMH standard curve diagram produced by the present invention.
[0034] Figure 2 This is the FSH standard curve diagram produced by the present invention. DETAILED DESCRIPTION
[0035] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.
[0036] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
[0037] Example 1
[0038] Preparation of test strips for combined detection of AMH and FSH:
[0039] 1. Solution Preparation
[0040] (1) Conjugate pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, 1 g of BSA, and 0.1 g of PEG20000 using a balance. Use a graduated cylinder to measure 4 mL of 50 mM boric acid buffer, dissolve in water, and adjust the volume to 100 mL.
[0041] (2) Sample pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, and 1 g of BSA using a balance, adding 50 mM borate buffer to dissolve and then adjusting the volume to 100 mL.
[0042] (3) Coating antibody diluent: Prepare 10 mL of solution by weighing 0.1 g of sodium chloride and 0.2 g of trehalose using a balance, adding phosphate buffer (0.01 M PBS Ph7.2-7.4) to dissolve and then adjust the volume to 10 mL.
[0043] 2. Pretreatment of conjugate pad and sample pad
[0044] Cut the glass cellulose membrane into sample pads and conjugate pads according to the specifications of 17*300mm and 8*300mm, respectively, soak them in the sample pad pretreatment solution and conjugate pad pretreatment solution for 20 minutes, then put them in a drying oven at 40℃ for 3 hours, and then put them in a dry place for use.
[0045] 3. Fluorescent microsphere labeled antibodies (antibody labeling solution)
[0046] Remove the fluorescent microspheres, centrifuge at 13000 rpm / min and discard the supernatant, add phosphate buffer (pH6.0-7.0) to adjust the microsphere concentration to 1%, add EDC and NHS with a final concentration of 2 mg / mL, and activate at room temperature for 30 minutes. Rotate the reaction at room temperature for 1.5 hours, centrifuge at 13000 rpm / min and discard the supernatant, then dissolve with phosphate buffer (pH6.0-7.0), and ultrasonically disperse the re-dissolved microspheres. (1) Add human AMH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human AMH monoclonal antibody 1 labeling solution. (2) Add human FSH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human FSH monoclonal antibody 1 labeling solution. (3) Add human AMH polyclonal antibody at 0.2 mg / mL for labeling to prepare human AMH polyclonal antibody labeling solution. (4) Add human FSH polyclonal antibody at 0.2 mg / mL for labeling to prepare human FSH polyclonal antibody labeling solution.
[0047] 4. Combined pad spraying
[0048] Remove the pretreated conjugate pad and the prepared fluorescent microsphere-labeled antibody. First, mix human AMH monoclonal antibody 1 labeling solution, human FSH monoclonal antibody 1 labeling solution, human AMH polyclonal antibody labeling solution, and human FSH polyclonal antibody labeling solution in a ratio of 1:1:0.5:0.5 to prepare a fluorescent microsphere-labeled antibody mixture. Use an XYZ gold sprayer to spray the pretreated, dried conjugate pad at 6 μL / cm. Place the conjugate pad in a 40°C drying oven to dry for 3 hours. After drying, place it in a dry place for later use.
[0049] 5. Streaking of Nitrocellulose Membrane
[0050] First, use the coating antibody diluent to dilute AMH monoclonal antibody 2, FSH monoclonal antibody 2 and goat anti-mouse IgG to 1.0 mg / mL, 1.0 mg / mL and 0.8 mg / mL, respectively. Then use an XYZ gold spray film streak instrument to streak on the nitrocellulose membrane at 1 μL / cm. Place the nitrocellulose membrane in a 40°C drying oven and dry it for 3 hours. After drying, put it in a dry place for use.
[0051] 6. Test strip assembly
[0052] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were sequentially pasted on the PVC base plate and cut into 3.00 mm wide test strips using a fully automatic chopping machine.
[0053] Example 2
[0054] Preparation of test strips for combined detection of AMH and FSH:
[0055] 1. Solution Preparation
[0056] (1) Conjugate pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, 1 g of BSA, and 0.1 g of PEG20000 using a balance. Use a graduated cylinder to measure 4 mL of 50 mM boric acid buffer, dissolve in water, and adjust the volume to 100 mL.
[0057] (2) Sample pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, and 1 g of BSA using a balance, adding 50 mM borate buffer to dissolve and then adjusting the volume to 100 mL.
[0058] (3) Coating antibody diluent: Prepare 10 mL of solution by weighing 0.1 g of sodium chloride and 0.2 g of trehalose using a balance, adding phosphate buffer (0.01 M PBS Ph7.2-7.4) to dissolve and then adjust the volume to 10 mL.
[0059] 2. Pretreatment of conjugate pad and sample pad
[0060] Cut the glass cellulose membrane into sample pads and conjugate pads according to the specifications of 17*300mm and 8*300mm, respectively, soak them in the sample pad pretreatment solution and conjugate pad pretreatment solution for 20 minutes, then put them in a drying oven at 40℃ for 3 hours, and then put them in a dry place for use.
[0061] 3. Fluorescent microsphere labeled antibodies (antibody labeling solution)
[0062] Remove the fluorescent microspheres, centrifuge at 13000 rpm / min and discard the supernatant, add phosphate buffer (pH 6.0-7.0) to adjust the microsphere concentration to 0.1%, add EDC and NHS with a final concentration of 5 mg / mL, and activate at room temperature for 40 minutes. Rotate and react at room temperature for 3 hours, centrifuge at 13000 rpm / min and discard the supernatant, then dissolve with phosphate buffer (pH 6.0-7.0), and ultrasonically disperse the re-dissolved microspheres. (1) Add human AMH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human AMH monoclonal antibody 1 labeling solution. (2) Add human FSH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human FSH monoclonal antibody 1 labeling solution. (3) Add human AMH polyclonal antibody at 0.2 mg / mL for labeling to prepare human AMH polyclonal antibody labeling solution. (4) Add human FSH polyclonal antibody at 0.2 mg / mL for labeling to prepare human FSH polyclonal antibody labeling solution.
[0063] 4. Combined pad spraying
[0064] Remove the pretreated conjugate pad and the prepared fluorescent microsphere-labeled antibody. First, mix human AMH monoclonal antibody 1 labeling solution, human FSH monoclonal antibody 1 labeling solution, human AMH polyclonal antibody labeling solution, and human FSH polyclonal antibody labeling solution in a ratio of 1:1:0.2:0.2 to prepare a fluorescent microsphere-labeled antibody mixture. Use an XYZ gold sprayer to spray the pretreated, dried conjugate pad at 6 μL / cm. Place the conjugate pad in a 40°C drying oven to dry for 3 hours. After drying, store it in a dry place until ready for use.
[0065] 5. Streaking of Nitrocellulose Membrane
[0066] First, use the coating antibody diluent to dilute AMH monoclonal antibody 2, FSH monoclonal antibody 2 and goat anti-mouse IgG to 0.5 mg / mL, 0.5 mg / mL and 0.3 mg / mL, respectively. Then use an XYZ gold spray film streak instrument to streak on the nitrocellulose membrane at 0.8 μL / cm. Place the nitrocellulose membrane in a 40°C drying oven and dry it for 3 hours. After drying, put it in a dry place for use.
[0067] 6. Test strip assembly
[0068] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were sequentially pasted on the PVC base plate and cut into 3.00 mm wide test strips using a fully automatic chopping machine.
[0069] Example 3
[0070] Preparation of test strips for combined detection of AMH and FSH:
[0071] 1. Solution Preparation
[0072] (1) Conjugate pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, 1 g of BSA, and 0.1 g of PEG20000 using a balance. Use a graduated cylinder to measure 4 mL of 50 mM boric acid buffer, dissolve in water, and adjust the volume to 100 mL.
[0073] (2) Sample pad pretreatment solution: Prepare 100 mL of solution by weighing 3 g of trehalose, 0.2 g of Tween-20, and 1 g of BSA using a balance, adding 50 mM borate buffer to dissolve and then adjusting the volume to 100 mL.
[0074] (3) Coating antibody diluent: Prepare 10 mL of solution by weighing 0.1 g of sodium chloride and 0.2 g of trehalose using a balance, adding phosphate buffer (0.01 M PBS Ph7.2-7.4) to dissolve and then adjust the volume to 10 mL.
[0075] 2. Pretreatment of conjugate pad and sample pad
[0076] Cut the glass cellulose membrane into sample pads and conjugate pads according to the specifications of 17*300mm and 8*300mm, respectively, soak them in the sample pad pretreatment solution and conjugate pad pretreatment solution for 20 minutes, then put them in a drying oven at 40℃ for 3 hours, and then put them in a dry place for use.
[0077] 3. Fluorescent microsphere labeled antibodies (antibody labeling solution)
[0078] Remove the fluorescent microspheres, centrifuge at 13000 rpm / min and discard the supernatant, add phosphate buffer (pH 6.0-7.0) to adjust the microsphere concentration to 0.5%, add EDC and NHS with a final concentration of 0.1 mg / mL, and activate at room temperature for 20 minutes. Rotate and react at room temperature for 2 hours, centrifuge at 13000 rpm / min and discard the supernatant, then dissolve with phosphate buffer (pH 6.0-7.0), and ultrasonically disperse the re-dissolved microspheres. (1) Add human AMH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human AMH monoclonal antibody 1 labeling solution. (2) Add human FSH monoclonal antibody 1 at 0.5 mg / mL for labeling to prepare human FSH monoclonal antibody 1 labeling solution. (3) Add human AMH polyclonal antibody at 0.2 mg / mL for labeling to prepare human AMH polyclonal antibody labeling solution. (4) Add human FSH polyclonal antibody at 0.2 mg / mL for labeling to prepare human FSH polyclonal antibody labeling solution.
[0079] 4. Combined pad spraying
[0080] Remove the pretreated conjugate pad and the prepared fluorescent microsphere-labeled antibody. First, mix human AMH monoclonal antibody 1 labeling solution, human FSH monoclonal antibody 1 labeling solution, human AMH polyclonal antibody labeling solution, and human FSH polyclonal antibody labeling solution in a ratio of 1:1:0.3:0.3 to prepare a fluorescent microsphere-labeled antibody mixture. Use an XYZ gold sprayer to spray the pretreated, dried conjugate pad at 6 μL / cm. Place the conjugate pad in a 40°C drying oven to dry for 3 hours. After drying, store it in a dry place until ready for use.
[0081] 5. Streaking of Nitrocellulose Membrane
[0082] First, use the coating antibody diluent to dilute AMH monoclonal antibody 2, FSH monoclonal antibody 2 and goat anti-mouse IgG to 0.8 mg / mL, 0.8 mg / mL and 0.5 mg / mL, respectively. Then use an XYZ gold spray film streak instrument to streak on the nitrocellulose membrane at 0.9 μL / cm. Place the nitrocellulose membrane in a 40°C drying oven and dry it for 3 hours. After drying, put it in a dry place for use.
[0083] 6. Test strip assembly
[0084] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were sequentially pasted on the PVC base plate and cut into 3.00 mm wide test strips using a fully automatic chopping machine.
[0085] Example 4
[0086] Standard curve preparation
[0087] (1) Preparation of standard solution:
[0088] 1) AMH standard solution: Use human AMH-negative blood to dilute the purchased AMH standard to a concentration of 0 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, and 25 ng / mL.
[0089] 2) FSH standard solution: Use human FSH-negative blood to dilute the FSH national standard to obtain sample solutions with dilution concentrations of 200mIU / mL, 150mIU / mL, 100mIU / mL, 75mIU / mL, 50mIU / mL, 20mIU / mL, 15mIU / mL, 10mIU / mL, 5mIU / mL, and 0mIU / mL.
[0090] (2) Fluorescence value reading: AMH and FSH sample solutions were tested on the reagent card respectively. After adding the samples, the card was placed in the fluorescence immunoassay analyzer. After 10 minutes, the fluorescence values of the detection lines T1, T2 and C were obtained. Each concentration was repeated three times and the average value was obtained.
[0091] (3) Establishment of standard curve: Based on the fluorescence intensity data of the test line and the quality control line, analyze the data, use the sample concentration as the horizontal axis and the fluorescence intensity ratio of the test line / quality control line (T1 / C or T2 / C) as the vertical axis, and fit it into a standard curve for quantitative detection of the sample concentration. Figure 1 and Figure 2 .
[0092] Example 5
[0093] Test strip performance testing
[0094] (1) Detection limit
[0095] 1) AMH Detection Limit: The test strips were tested with AMH standard solutions at concentrations of 0.05 ng / mL, 0.1 ng / mL, 0.15 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2 ng / mL, 5 ng / mL, and 10 ng / mL. Each concentration was tested three times. The test results were sorted by size, and the AMH concentration that just barely detected was defined as the detection limit. The test strips fully detected the 0.1 ng / mL standard solution, so the AMH detection limit was ≤ 0.1 ng / mL.
[0096] 2) FSH Detection Limit: Test the test strips with FSH standard solutions at concentrations of 20mIU / mL, 15mIU / mL, 10mIU / mL, 7.5mIU / mL, 5mIU / mL, and 2.5mIU / mL. Test each concentration three times, sort the test results by size, and define the FSH concentration that just barely detects it as the detection limit. The test strips can fully detect the 10mIU / mL standard solution, so the FSH detection limit is 10mIU / mL.
[0097] (2) Accuracy
[0098] 1) AMH: Based on the linear range, anti-Mullerian hormone standards were prepared at concentrations of 0.5 ng / ml, 2.5 ng / ml, and 4.0 ng / ml for testing. Each sample was assayed five times, and the measured values were compared with the theoretical values. As shown in Table 1 below, the relative deviation between the measured and theoretical values was within ±10%.
[0099] Table 1
[0100]
[0101] 2) FSH: Based on the linear range, 10mIU / ml, 25mIU / ml, and 50mIU / ml FSH standards were prepared for testing. Each sample was assayed five times, and the measured values were compared with the theoretical values. As shown in Table 2 below, the relative deviation between the measured and theoretical values was within ±10%.
[0102] Table 2
[0103]
[0104] (3) Specificity
[0105] The FSH test strips and AMH test strips were tested with 200mIU / mL LH, 100IU / mL rheumatoid factor (RF), 0.6mg / mL bilirubin, triglycerides and 0.5mg / mL hemoglobin respectively. The results showed that FSH was less than 1mIU / mL and AMH was less than 0.1ng / mL, indicating that there was no cross reaction between the FSH and AMH test strips and the above-mentioned interferents, indicating that the specificity of this test strip is strong.
[0106] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.
[0107] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.
Claims
1. A test strip for jointly detecting AMH and FSH, characterized in that, The method comprises a conjugate pad pretreated with a conjugate pad pretreatment solution, a sample pad pretreated with a sample pad pretreatment solution, and a nitrocellulose membrane. The nitrocellulose membrane is coated with an antibody diluted with a coating antibody diluent.
2. a kind of test strip of joint detection AMH and FSH as claimed in claim 1, is characterized in that: The conjugate pad pretreatment solution included 3% trehalose, 0.2% Tween-20, 1% BSA, 0.1% PEG20000 and 4% 50 mM borate buffer.
3. a kind of test strip of joint detection AMH and FSH as claimed in claim 2, is characterized in that: The conjugate pad pretreated with the conjugate pad pretreatment solution is also sprayed with an antibody mixture labeled with fluorescent microspheres, wherein the antibody mixture includes human AMH monoclonal antibody 1, human FSH monoclonal antibody 1, human AMH polyclonal antibody and human FSH polyclonal antibody.
4. a kind of test strip of joint detection AMH and FSH as claimed in claim 1, is characterized in that: The sample pad pretreatment liquid included 3% trehalose, 0.2% Tween-20, 1% BSA, and 50 mM borate buffer.
5. a kind of test strip of joint detection AMH and FSH as claimed in claim 1, is characterized in that: The antibodies coated on the nitrocellulose membrane include human AMH monoclonal antibody 2, human FSH monoclonal antibody 2 and goat anti-mouse IgG monoclonal antibody, and the coating antibody diluent includes phosphate buffer containing 1% sodium chloride and 2% trehalose.
6. as the preparation method of the test strip of a kind of joint detection AMH and FSH as described in any one of claim 1-5, it is characterized in that: include: Preparation of the conjugate pad: pre-treat the conjugate pad with a conjugate pad pre-treatment solution, dry it, and then spray it with a mixture of antibodies labeled with fluorescent microspheres; Preparation of sample pad: pre-treat the sample pad with sample pad pre-treatment solution and dry it for later use; Streak the nitrocellulose membrane: dilute the antibody with the coating antibody diluent, then streak the nitrocellulose membrane. After streaking, dry the nitrocellulose membrane for later use. Test strip assembly: stick the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence and cut into test strips.
7. a preparation method of the test strip of joint detection AMH and FSH as claimed in claim 6, is characterized in that: The conjugate pad was soaked in the conjugate pad pretreatment solution for 20 minutes, and then taken out and dried in a drying oven at 40° C. for 3 hours.
8. a preparation method of the test strip of a kind of joint detection AMH and FSH as claimed in claim 7, is characterized in that: The method for pretreating a dried conjugate pad and spraying a mixed antibody solution labeled with fluorescent microspheres includes: centrifuging the microspheres and discarding the supernatant, then adding phosphate buffer to adjust the microsphere concentration to 0.1-1%, adding EDC and NHS with a final concentration of 0.1-5 mg / mL, activating at room temperature for 20-40 minutes, rotating at room temperature for 1.5-3 hours, centrifuging and discarding the supernatant, then dissolving with phosphate buffer, ultrasonically dispersing the re-dissolved microspheres, and then adding human AMH monoclonal antibody 1, human FSH monoclonal antibody 1, human AMH polyclonal antibody and human FSH polyclonal antibody at a concentration of 0.1-1.0 mg / mL. Labeling was performed to obtain human AMH monoclonal antibody 1 labeling solution, human FSH monoclonal antibody 1 labeling solution, human AMH polyclonal antibody labeling solution and human FSH polyclonal antibody labeling solution. Human AMH monoclonal antibody 1 labeling solution, human FSH monoclonal antibody 1 labeling solution, human AMH polyclonal antibody labeling solution and human FSH polyclonal antibody labeling solution were mixed in a ratio of 1:1:(0.2-0.5):(0.2-0.5) to obtain an antibody mixture, which was then sprayed on a pretreated and dried conjugate pad at 6 μL / cm using an XYZ gold spray film apparatus, and finally dried in a drying oven at 40°C for 3 h.
9. a preparation method of the test strip of joint detection AMH and FSH as claimed in claim 6, is characterized in that: After the sample pad was soaked in the sample pad pretreatment solution for 20 minutes, it was taken out and dried in a drying oven at 40°C for 3 hours.
10. a preparation method of a test strip for jointly detecting AMH and FSH as claimed in claim 6, characterized in that: The concentration of human AMH monoclonal antibody 2 after dilution was 0.5-1 mg / mL, the concentration of human FSH monoclonal antibody 2 after dilution was 0.5-1 mg / mL, and the concentration of goat anti-mouse IgG monoclonal antibody after dilution was 0.3-0.8 mg / mL. Then, an XYZ gold spray film streak was used to streak the nitrocellulose membrane at 0.8-1 μL / cm. After streaking, the nitrocellulose membrane was placed in a drying oven at 40°C for 3 hours.