AMH semi-quantitative latex method detection kit and preparation method thereof

Abstract

The invention discloses an AMH semi-quantitative latex method detection kit and a preparation method thereof. The latex method detection kit comprises two test paper cards with different sensitivities. According to the present invention, the rapid semi-quantitative detection of the AMH level in the whole blood sample is achieved through the test paper cards with different sensitivities, the sampling field can be immediately analyzed without any equipment or other auxiliary reagents during the detection, the occupation of the laboratory resources can be effectively reduced, the operation is convenient, and the use is easy.

Description

Technical Field

[0001] The invention belongs to the field of latex method detection and relates to an AMH semi-quantitative latex method detection kit and a preparation method thereof. Background Art

[0002] Anti-Mullerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is a dimeric glycoprotein that belongs to the transforming growth factor β (TGF-β) and differentiation factor family. AMH mainly exerts its biological effects through AMH receptor Ⅱ. AMH in women is mainly produced by the granulosa cells of the preantral follicles and small antral follicles of the ovaries and secreted into the blood. It inhibits the recruitment of primordial follicles and the development of antral follicles, preventing premature exhaustion of follicles. The main function of AMH is to evaluate the ovarian reserve function of women, and it is a routine indicator for evaluating ovarian reserve.

[0003] Clinically, the AMH cutoff level for decreased ovarian reserve in women over 40 years old is defined as 0.5-1.1 μg / L. The 2016 Poseidon standard is based on the ESHRE definition of the AMH cutoff level for decreased ovarian reserve, and AMH < 1.2 μg / L is used as a predictor of poor ovarian response. The serum AMH level of patients with polycystic ovary syndrome (PCOS) is 2-3 times higher than that of normal women of the same age. The AMH level for the diagnosis of polycystic ovary syndrome (PCOM) is 20-27.14 pmol / L.

[0004] At present, most of the AMH detection methods in clinical practice include chemiluminescence, fluorescence immunochromatography, enzyme-linked immunosorbent assay, etc. Enzyme immunoassay is complicated to operate and takes a long time to detect; the detection equipment supporting fluorescence immunoassay is much cheaper than chemiluminescence, but it still cannot get rid of the limitations of the equipment. Therefore, there is an urgent need for a simple and operable semi-quantitative AMH detection method that does not require instruments. Summary of the invention

[0005] The technical problem solved by the present invention is to solve the defects of the prior art and provide an AMH semi-quantitative detection kit which is suitable for on-site diagnosis and home self-examination, is simple and convenient to operate, and does not require any other instruments when used.

[0006] One of the technical problems to be solved by the present invention is to provide a highly sensitive whole blood sample processing pad composed of an anti-Mullerian hormone test paper card, which can be analyzed on the sampling site without the need for laboratory testing. There is no need for complex processing and dilution of the whole blood sample, and no need for any equipment.

[0007] The second technical problem to be solved by the present invention is to optimize the detection method of AMH, which is simple to use and improves sensitivity on the basis of reducing costs, so as to confirm the content of AMH in the whole blood sample in a semi-quantitative manner.

[0008] The third technical problem to be solved by the present invention is to provide a method for using an anti-Mullerian hormone test paper card.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] On one hand, the present invention provides a latex method anti-Mullerian hormone detection kit, characterized in that it includes two groups of test paper cards with different sensitivities; the two groups of test paper cards with different sensitivities each include a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper;

[0011] The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are overlapped in sequence and pasted on the bottom plate;

[0012] The conjugate pad is sprayed with a latex microsphere conjugate solution; the latex microsphere conjugate is an AMH capture antibody and a rabbit IgG antibody labeled with latex microspheres.

[0013] A detection line and a quality control line are arranged in parallel from one end of the binding pad of the nitrocellulose membrane to one end of the absorbent paper, and the quality control line and the detection line are parallel to the overlap seam;

[0014] The quality control line and the detection line are sprayed with goat anti-rabbit IgG and AMH monoclonal antibody respectively;

[0015] The detection limits of the two groups of test strips with different sensitivities for anti-Mullerian hormone can be selected from the following groups:

[0016] i) 1 ng / mL and 10 ng / mL,

[0017] ii) 0.5 ng / mL and 7.5 ng / mL.

[0018] As a preference:

[0019] The expression sequence of the AMH capture antibody labeled with latex microspheres is shown in SEQ No. 1;

[0020] The expression sequence of AMH monoclonal antibody is shown in SEQ No.2.

[0021] The above-mentioned AMH capture antibody and AMH monoclonal antibody are both artificially synthesized, and their coding sequences are shown in SEQ No. 1 and SEQ No. 2, and recombinant proteins are obtained after expression.

[0022] Preferably, in the latex microsphere conjugate solutions sprayed on the two groups of test strips with different sensitivities, the concentrations of the AMH capture antibodies labeled with the latex microspheres are different, and the concentration of the AMH capture antibodies is selected from the following group according to the sensitivity requirements:

[0023] i) 5-25 μg / mL and 0.5-5 μg / mL, preferably 5 μg / mL and 0.5 μg / mL

[0024] ii) 5 to 25 μg / mL and 0.5 to 3.75 μg / mL, preferably 10 μg / mL and 3.75 μg / mL.

[0025] Preferably, in the latex microsphere conjugate solutions sprayed on the two groups of test strips of different sensitivities, the concentrations of rabbit IgG antibodies labeled with latex microspheres are the same, and the rabbit IgG antibody mass concentration is 5 to 25 μg / mL, preferably 5 μg / mL.

[0026] Preferably, the average particle size of the latex microsphere particles is 100-600 nm, preferably 400 nm.

[0027] Preferably, the concentration of AMH monoclonal antibody and goat anti-rabbit IgG is 1 mg / mL, and the spraying amount is 0.8 uL / cm. The antibodies are sprayed on the nitrocellulose membrane to form a detection line and a quality control line with a spacing of 5 mm. After drying, the membrane is sealed for later use.

[0028] Preferably, the binding pad is selected from glass fiber membrane; the pretreatment method is to soak the glass fiber membrane in 20 mmol Tris buffer with a pH of 7.4 containing 0.05% Tween-20, 1% bovine serum albumin, and 2% trehalose, dry it at room temperature for 10 minutes, and then dry it at 37°C for 4 hours.

[0029] Preferably, the sample pad is pretreated by soaking the sample pad in a 20 mmol Tris buffer at pH 7.4 containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein, and 500 ng to 5 mg / mL lectin, drying at room temperature for 10 minutes, and then drying at 37°C for 4 hours.

[0030] Preferably, the kit further comprises a sample diluent; the sample diluent is an aqueous solution of a mixture of sodium chloride, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, potassium chloride and Triton.

[0031] Preferably, the concentrations of the substances are:

[0032] Sodium chloride 8g / L;

[0033] Disodium hydrogen phosphate dodecahydrate 0.126 g / L;

[0034] Potassium dihydrogen phosphate 0.06g / L;

[0035] Potassium chloride 0.4 g / L;

[0036] Triton 0.01% / L.

[0037] In some specific embodiments, the present invention designs two different test kits, A and B, for the reference range of AMH concentration in women of different age groups. Among them, the AMH detection range of type A is 1-10ng / mL, which is suitable for women aged 18-29 years old; the AMH detection range of type B is 0.5-7.5ng / mL, which is suitable for women aged 30-40 years old.

[0038] Furthermore, Type A and Type B respectively include two test paper cards with different sensitivities, and their product compositions are specifically shown below.

[0039]

[0040] In some specific embodiments, rabbit IgG antibody and goat anti-rabbit IgG polyclonal antibody are purchased finished antibodies, both from Xibao Biotechnology. The rabbit IgG antibody item number is EXK3226B; goat anti-rabbit IgG is ECY4491A.

[0041] The latex microsphere immunochromatographic test paper card adopts a double antibody sandwich immunoassay.

[0042] The present invention distinguishes the sensitivity by capturing AMH of different concentrations by coating AMH capture antibodies of different concentrations. When AMH is contained in the blood, the AMH in the blood combines with the AMH capture antibody labeled with latex microspheres to form a complex, and the complex moves forward along the paper strip under the action of chromatography, and reacts with the pre-coated anti-AMH monoclonal antibody when passing through the detection line to form an immune complex and present a red strip, and the free latex microsphere-labeled rabbit IgG antibody combines with the goat anti-rabbit antibody on the quality control line to present a red strip. Which test paper cards develop color is determined by the content of AMH in the blood.

[0043] Interpretation of results

[0044] Type A test kit is suitable for women aged 18-29, and they need to use type A card 1 and type A card 2 for testing respectively. When the quality control line C of type A card 1 and type A card 2 are both colored normally; if type A card 1 is not detected, it means that the AMH level is lower than 1ng / mL, indicating that the woman's ovarian reserve function is poor and there may be a risk of premature ovarian failure; if type A card 2 is detected, it indicates that the woman's AMH level is too high and there may be a risk of polycystic ovary syndrome; if type A card 1 is detected but type A card 2 is not detected, the woman's ovarian reserve function is between 1 and 10ng / mL, which is a normal level.

[0045] The B-type test kit is suitable for women aged 30-40, and they need to be tested using B-type card 1 and B-type card 2 respectively. When the quality control line C of B-type card 1 and B-type card 2 are normal, but B-type card 1 is not detected, it means that the AMH level is lower than 0.5ng / mL, indicating that the woman's ovarian reserve function is poor and there may be a risk of premature ovarian failure; if B-type card 2 is detected, it indicates that the woman's AMH level is too high and there may be a risk of polycystic ovary syndrome; if B-type card 1 is detected but B-type card 2 is not detected, the woman's ovarian reserve function is between 0.5 and 7.5ng / mL, which is a normal level.

[0046] The present invention has the following beneficial effects:

[0047] The present invention can efficiently detect AMH in the blood through specially designed AMH capture antibodies and AMH monoclonal antibodies. The detection kit of the present invention can be used for semi-quantitative self-testing of the content of anti-Mullerian hormone (AMH) in human blood samples in vitro, with simple operation and accurate result judgment. The self-test at home can examine the ovarian reserve function of the individual and assist in the diagnosis of polycystic ovary syndrome. The present invention is also conducive to the self-assessment of the body by women in the period of preparing for pregnancy, indicating the possibility of polycystic ovary syndrome; it can also be used for the estimation and maintenance of menopause in elderly women, reducing the risk of osteoporosis and delaying cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the structure of the AMH test paper card: 1 is absorbent paper, 2 is nitrocellulose membrane, 3 is binding pad, 4 is sample pad, 5 is bottom plate, T is the detection line membrane position, and C is the quality control line membrane position.

[0049] Figure 2 Schematic diagram of the AMH test paper cards included in the Type A and Type B AMH detection kits: 6 and 8 are quality control lines for Type A cards, 7 is the T line for Type A card 1 detection (1 ng / mL), 9 is the T line for Type A card 2 detection (10 ng / mL), 10 and 12 are quality control lines for Type B cards, 11 is the T line for Type B card 1 detection (0.5 ng / mL), 13 is the T line for Type B card 2 detection (7.5 ng / mL), and 14 is the sample addition hole. DETAILED DESCRIPTION

[0050] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0051] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0052] Figure 1The present invention shows an anti-Mullerian hormone (AMH) test paper card, comprising a base plate 5, on which a sample pad 4, a conjugation pad 3, a nitrocellulose membrane 2, and a water-absorbing paper 1 are sequentially attached; one end of the conjugation pad 3 overlaps with the sample pad 4, and the other end overlaps with one end of the nitrocellulose membrane 2, and the other end of the nitrocellulose membrane 2 overlaps with the water-absorbing paper 1; a detection line T and a quality control line C are arranged on the nitrocellulose membrane 2; the detection line T has an AMH monoclonal antibody in the solid phase; and the quality control line C is coated with goat anti-rabbit IgG. A latex microsphere complex is sprayed on the conjugation pad 3.

[0053] The anti-Mullerian hormone (AMH) test kit disclosed in the present invention is divided into type A and type B for women of different age groups. Each type of test kit includes two groups of AMH test paper cards with different detection line sensitivities, and a latex microsphere complex is sprayed on the binding pad of each group of AMH test paper cards. The latex microsphere complex includes an AMH capture antibody and a rabbit IgG antibody labeled with latex microspheres. Among them, the concentrations of the AMH capture antibodies labeled with latex microspheres sprayed on the AMH test paper cards of different sensitivities are different.

[0054] In the type A AMH test kit for women aged 18-29, the two sets of AMH test strips had detection sensitivities of 1 ng / mL and 10 ng / mL for AMH, respectively, and the corresponding concentrations of AMH capture antibodies labeled with sprayed latex microspheres were 5 μg / mL and 0.5 μg / mL, respectively.

[0055] In the type B AMH test kit for women aged 30-39, the detection sensitivities of the two sets of AMH test strips for AMH were 0.5 ng / mL and 7.5 ng / mL, respectively, and the corresponding concentrations of the sprayed latex microsphere-labeled AMH capture antibodies were 10 μg / mL and 3.75 μg / mL, respectively.

[0056] The concentration of rabbit IgG antibody labeled with latex microspheres sprayed on all AMH test strips is 5μg / mL.

[0057] The above-mentioned latex microsphere-labeled AMH capture antibody and rabbit IgG antibody were both activated using conventional EDC.

[0058] Wherein, the nucleotide coding sequence of the AMH capture antibody labeled with latex microspheres is shown in SEQ No.1;

[0059] The nucleotide coding sequence of the AMH monoclonal antibody is shown in SEQ No.2:

[0060] Rabbit IgG antibody and goat anti-rabbit IgG polyclonal antibody are purchased finished antibodies, both from Xibao Biotechnology. The rabbit IgG antibody is EXK3226B; goat anti-rabbit IgG is ECY4491A.

[0061] Example 1

[0062] 1. Pretreatment of the conjugate pad

[0063] At room temperature, the glass fiber membrane was soaked in 20 mmol Tris buffer (pH 7.4) containing 0.05% Tween-20, 1% bovine serum albumin, and 2% trehalose, dried for 10 minutes, and then dried at 37°C for 4 hours. It was dried and sealed for use to prepare a conjugate pad for subsequent gold spraying.

[0064] 2. Preparation of coating solution

[0065] Anti-AMH monoclonal antibody and goat anti-rabbit IgG antibody were diluted to 1 mg / mL respectively using PB buffer containing 0.9% NaCl + 3% sucrose 10 mM, and streaked on a nitrocellulose membrane at 0.1 μL / mm using a gold spray film streaker.

[0066] 3. Sample Pad Pretreatment

[0067] The sample pad was soaked in 20 mmol Tris buffer (pH 7.4) containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein, and 500 ng to 5 mg / mL lectin, and dried at room temperature for 10 minutes, then dried at 37°C for 4 hours, dried and sealed for later use.

[0068] 4. Latex microsphere labeling

[0069] (1) Using the conventional EDC method, latex microspheres were activated in 50 mM MES buffer for half an hour;

[0070] (2) Centrifuge at 12000g for 10 min and remove the supernatant;

[0071] (3) 20ul of activated latex microspheres (excess, average particle size 400nm) were resuspended in 1mL (50mM, pH 8.0) of PB buffer, and AMH capture antibody (different test strips have different amounts (specific addition amounts are shown in Table 1) or rabbit IgG antibody (1μg) was added. After incubation for 1 hour, 20% bovine serum albumin solution was added as blocking solution and the reaction was allowed to proceed for 30 minutes.

[0072] Table 1

[0073]

[0074]

[0075] (4) The latex microsphere solution obtained in step (3) was centrifuged at 4°C and 12000rpm for 20min, the supernatant was removed after centrifugation, and the precipitate was redissolved with a resolution solution, wherein the resolution solution was a PB buffer solution containing 0.05% Tween 20 and 0.1% BSA, and the amount was 100uL, so that the AMH capture antibody labeled with latex microspheres and the rabbit IgG antibody labeled with latex microspheres were obtained respectively. The two antibodies labeled with latex microspheres were mixed in a volume ratio of 1:1 to obtain a latex microsphere complex, wherein the concentration of the AMH capture antibody is shown in Table 1, and the concentration of the rabbit IgG antibody labeled with latex microspheres is 5μg / mL (based on the mass concentration of the rabbit IgG antibody).

[0076] (5) spraying the latex microsphere complex obtained in step (4) onto the conjugate pad at a rate of 0.1 μL / mm using a gold spray film scribing instrument;

[0077] (6) The conjugate pad was dried in an oven at 37°C for 240 min. After drying, the pad was sealed with a desiccant and stored.

[0078] 5. Assembly and board mounting

[0079] The absorbent paper, nitrocellulose membrane, conjugate pad and sample pad are assembled on the bottom plate in sequence, and then cut with a high-speed strip cutter to obtain the finished AMH test paper card.

[0080] AMH test kits are divided into type A and type B for women of different age groups. Each type of test kit includes two sets of AMH test strips with different detection line sensitivities. Figure 2 As shown, the type A AMH detection kit includes type A card 1 and type A card 2, and the type B AMH detection kit includes type B card 1 and type B card 2.

[0081] In the present invention, the method of using the test paper card is as follows: take 1-2 drops (about 15-25 μL) of whole blood sample and slowly drip it into the sample addition hole of the test paper card, then add 2-3 drops (about 35-45 μL) of sample diluent and read the result after 15 minutes. The result may be abnormal after 15 minutes.

[0082] The sample diluent is: sodium chloride (8 g / L), disodium hydrogen phosphate dodecahydrate (0.126 g / L), potassium dihydrogen phosphate (0.06 g / L), potassium chloride (0.4 g / L), and Triton 0.01% / L.

[0083] Type A test kit is suitable for women aged 18-29, and they need to use type A card 1 and type A card 2 for testing respectively. When the quality control line C of type A card 1 and type A card 2 are both colored normally; if type A card 1 is not detected, it means that the AMH level is lower than 1ng / mL, indicating that the woman's ovarian reserve function is poor and there may be a risk of premature ovarian failure; if type A card 2 is detected, it indicates that the woman's AMH level is too high and there may be a risk of polycystic ovary syndrome; if type A card 1 is detected but type A card 2 is not detected, the woman's ovarian reserve function is between 1 and 10ng / mL, which is a normal level.

[0084] The B-type test kit is suitable for women aged 30-40, and they need to be tested using B-type card 1 and B-type card 2 respectively. When the quality control line C of B-type card 1 and B-type card 2 are normal, but B-type card 1 is not detected, it means that the AMH level is lower than 0.5ng / mL, indicating that the woman's ovarian reserve function is poor and there may be a risk of premature ovarian failure; if B-type card 2 is detected, it indicates that the woman's AMH level is too high and there may be a risk of polycystic ovary syndrome; if B-type card 1 is detected but B-type card 2 is not detected, the woman's ovarian reserve function is between 0.5 and 7.5ng / mL, which is a normal level.

[0085] If the quality control line of any test strip card does not have a red strip, it may be due to incorrect operation or the reagent has expired. The test result is invalid at this time and should be retested.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For the technical personnel in this field, the present invention can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made to the present invention should be included in the protection scope of the present invention.

Claims

1. A latex method anti-Mullerian hormone detection kit, characterized in that: The invention comprises two groups of test paper cards with different sensitivities; the two groups of test paper cards with different sensitivities each comprise a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper; The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are overlapped in sequence and pasted on the bottom plate; The conjugate pad is sprayed with a latex microsphere conjugate solution; the latex microsphere conjugate is an AMH capture antibody and a rabbit IgG antibody labeled with latex microspheres; A detection line and a quality control line are arranged in parallel from one end of the binding pad of the nitrocellulose membrane to one end of the absorbent paper, and the quality control line and the detection line are parallel to the overlap seam; The quality control line and the detection line are sprayed with goat anti-rabbit IgG and AMH monoclonal antibody respectively; the detection limit of the two groups of test strips with different sensitivities for anti-Mullerian hormone can be selected from the following groups: i) 1 ng / mL and 10 ng / mL, ii) 0.5 ng / mL and 7.5 ng / mL.

2. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The nucleotide coding sequence of the AMH capture antibody labeled with latex microspheres is shown in SEQ No.1; the nucleotide coding sequence of the AMH monoclonal antibody is shown in SEQ No.

2.

3. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The two groups of test strips with different sensitivities have different concentrations of AMH capture antibodies labeled with latex microspheres, and are selected from the following group according to the sensitivity requirements based on the mass concentration of AMH capture antibodies: i) 5-25 μg / mL and 0.5-5 μg / mL, preferably 5 μg / mL and 0.5 μg / mL ii) 5 to 25 μg / mL and 0.5 to 3.75 μg / mL, preferably 10 μg / mL and 3.75 μg / mL.

4. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The concentration of the rabbit IgG antibody labeled with latex microspheres sprayed on the two groups of test paper cards with different sensitivities is the same, which is 5 to 25 μg / mL, preferably 5 μg / mL, based on the mass concentration of the rabbit IgG antibody.

5. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The average particle size of the latex microsphere particles is 100-600 nm, preferably 400 nm.

6. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The concentration of AMH monoclonal antibody and goat anti-rabbit IgG is 1 mg / mL, and the spraying amount is 0.8 uL / cm. Spray the antibodies on the nitrocellulose membrane to form a detection line and a quality control line with a spacing of 5 mm. After drying, seal and set aside.

7. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The conjugate pad is selected from glass fiber membrane; the pretreatment method is to soak the glass fiber membrane in 20mmol Tris buffer with pH 7.4 containing 0.05% Tween-20, 1% bovine serum albumin, and 2% trehalose, dry it at room temperature for 10 minutes, and then dry it at 37°C for 4 hours.

8. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: The sample pad pretreatment method is as follows: soak the sample pad in a 20 mmol Tris buffer with a pH of 7.4 containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein, and 500 ng to 5 mg / mL lectin, dry it at room temperature for 10 minutes, and then dry it at 37°C for 4 hours.

9. The latex method anti-Mullerian hormone detection kit according to claim 1, characterized in that: It also includes a sample diluent; the sample diluent is an aqueous solution of a mixture of sodium chloride, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, potassium chloride and Triton.

10. The latex method anti-Mullerian hormone detection kit according to claim 9, characterized in that: The concentrations of each substance are:

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