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

Abstract

An AMH semi-quantitative latex-method detection kit, comprising two test strips having different sensitivities. Each test strip comprises a backing plate (5), a sample pad (4), a conjugate pad (3), a nitrocellulose membrane (2), and an absorbent pad (1). The conjugate pad (3) is sprayed with a latex microsphere conjugate solution, the latex microsphere conjugate comprising latex microsphere-labeled AMH capture antibodies and rabbit IgG antibodies. The nitrocellulose membrane (2) is provided with a test line and a control line, the control line and the test line being respectively sprayed with goat anti-rabbit IgG and AMH monoclonal antibodies. The test strips having different sensitivities achieve rapid semi-quantitative detection of AMH levels in whole blood samples without any equipment or other auxiliary reagents. Analysis can be performed immediately at the sampling site, enabling convenient operation.

Description

An AMH semi-quantitative latex detection kit and a preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of latex detection, and relates to an AMH semi-quantitative latex detection kit and a preparation method thereof. BACKGROUND

[0002] Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a dimeric glycoprotein belonging to the transforming growth factor beta (TGF-beta) and differentiation factor family. AMH mainly exerts biological effects through AMH receptor II (AMH receptor II). AMH in the female body is mainly produced and secreted into the blood by granulosa cells of preantral follicles and small antral follicles in the ovary, and has the effects of inhibiting the recruitment of primordial follicles and the development of antral follicles and preventing premature depletion of follicles. The main role of AMH is to evaluate the ovarian reserve function of women, and it is a conventional indicator for evaluating ovarian reserve.

[0003] The AMH cutoff level for the ovarian reserve function of women over 40 years old is defined as 0.5-1.1 μg / L in clinical practice, and the AMH cutoff level for the ovarian reserve function of women over 40 years old is defined as 0.5-1.1 μg / L in clinical practice. In 2016, the Poseidon standard is based on the definition of the AMH cutoff level for the ovarian reserve function of women over 40 years old by ESHRE, 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, and the AMH level for diagnosing polycystic ovary syndrome (PCOM) is 20-27.14 pmol / L.

[0004] At present, most of the AMH detection methods in clinical practice are chemiluminescence, fluorescence immunoassay, enzyme-linked immunoassay, etc. Enzyme immunoassay is complex to operate and takes a long time to detect; the detection equipment matched with fluorescence immunology is relatively cheap compared with chemiluminescence, but it is still limited by equipment. Therefore, there is an urgent need for an AMH semi-quantitative detection method without any equipment, which is simple and easy to operate. SUMMARY

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

[0006] One of the technical problems solved by the present application is to provide an anti-Mullerian hormone test paper card composed of a highly sensitive whole blood sample processing pad, which can be analyzed on site without laboratory testing. The whole blood sample does not need to be complicatedly processed and diluted, and does not require any equipment.

[0007] The second technical problem to be solved by the application is to optimize the detection method of AMH, which is simple to use, reduces the cost, and improves the sensitivity, so as to confirm the content of AMH in the whole blood sample by a semi-quantitative method.

[0008] The third technical problem to be solved by the application is to provide a use method of the anti-Mullerian hormone test paper card.

[0009] To achieve the above-mentioned purposes, the application provides the following technical solutions.

[0010] In one aspect, the application provides a latex anti-Mullerian hormone detection box, characterized in that the latex anti-Mullerian hormone detection box comprises two groups of test paper cards with different sensitivities.

[0011] The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent paper are sequentially spliced and pasted on the bottom plate.

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

[0013] The quality control line and the detection line are sequentially and parallelly arranged from one end of the conjugate 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 splicing seam.

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

[0015] The detection limit of the two groups of test paper cards for the anti-Mullerian hormone is optionally one of 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 preferred:

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

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

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

[0022] As a preferred, in the latex microsphere conjugate solution sprayed on the test paper card of the two groups of different sensitivity, the concentration of the latex microsphere labeled AMH capture antibody is different, and the concentration of the AMH capture antibody is selected from the following group according to the need of sensitivity:

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

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

[0025] As a preferred, in the latex microsphere conjugate solution sprayed on the test paper card of the two groups of different sensitivity, the concentration of the latex microsphere labeled AMH capture antibody is different, and the concentration of the AMH capture antibody is selected from the following group according to the need of sensitivity:

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

[0027] As a preferred, the concentration of the AMH monoclonal antibody and the goat anti-rabbit IgG is 1 mg / mL, the spraying amount is 0.8 uL / cm, and the antibodies are sprayed on the nitrocellulose membrane to form a detection line and a quality control line, the interval between the two lines is 5 mm, and after drying, it is sealed for standby.

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

[0029] As a preferred, the pretreatment method of the sample pad is to immerse the sample pad in a 20 mmol Tris buffer solution containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein, 500 ng-5 mg / mL phytohemagglutinin, dry at room temperature for 10 minutes, and then dry at 37°C for 4 hours.

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

[0031] As a preferred, the concentration of each substance is respectively:

[0032] In some specific embodiments, the AMH concentration reference range in the female body of different age groups is designed into two different detection boxes of A and B types. The AMH detection range of the A type is 1-10 ng / mL, which is suitable for 18-29 year-old women; the AMH detection range of the B type is 0.5-7.5 ng / mL, which is suitable for 30-40 year-old women.

[0033] Further, the A and B types respectively include two different sensitivity test paper cards, and the product composition is specifically as follows.

[0034] In some specific embodiments, the rabbit IgG antibody and the goat anti-rabbit IgG polyclonal antibody are commercially available finished antibodies, and are both from Xibao Biology. The rabbit IgG antibody is EXK3226B, and the goat anti-rabbit IgG is ECY4491A.

[0035] The latex microsphere immunochromatography test paper card adopts a double-antibody sandwich immunization method.

[0036] The application distinguishes the sensitivity by coating different concentrations of AMH capture antibodies to capture different concentrations of AMH. When the blood contains AMH, the AMH in the blood is combined with the latex microsphere labeled AMH capture antibody to form a complex, which moves forward along the paper strip under the chromatography effect, reacts with the pre-coated anti-AMH monoclonal antibody when passing through the detection line, forms an immune complex and presents a red band, and the free latex microsphere labeled rabbit IgG antibody is combined with the goat anti-rabbit antibody in the quality control line to present a red band. The content of AMH in the blood determines which test paper card develops color.

[0037] Explanation of results

[0038] The 18-29 year-old women use the A type detection box, and need to test by using the A type card 1 and the A type card 2 respectively. In the case that the quality control lines C of the A type card 1 and the A type card 2 are normally colored; the A type card 1 is not detected, which indicates that the AMH level is lower than 1 ng / mL, and it is prompted that the female has poor ovarian reserve function and may have the risk of premature ovarian failure; if the A type card 2 is detected, it is prompted that the AMH level of the female is too high, and there may be the risk of polycystic ovary syndrome; the A type card 1 is detected but the A type card 2 is not detected, which indicates that the ovarian reserve function of the female is between 1-10 ng / mL, and belongs to the normal level.

[0039] 30-40 years old female is suitable for B type detection box, and needs to use B type card 1 and B type card 2 respectively for testing. In the case that the quality control line C of B type card 1 and B type card 2 is normal, B type card 1 is not detected, it is indicated that the AMH level is lower than 0.5 ng / mL, and it is prompted that the female has poor ovarian reserve function, and there is a risk of premature ovarian failure; if B type card 2 is detected, it is prompted that the AMH level of the female is too high, and there is a risk of polycystic ovary syndrome; B type card 1 is detected but B type card 2 is not detected, the ovarian reserve function of the female is between 0.5-7.5 ng / mL, and it belongs to normal level.

[0040] The present application has the following beneficial effects:

[0041] The present application can be used for detecting the content of anti-Mullerian hormone (AMH) in human blood sample in vitro semi-quantitatively, and has the advantages of simple operation, accurate result judgment, self-test of ovarian reserve function at home, auxiliary diagnosis of polycystic ovary syndrome, self-evaluation of body of female in preparation period, prompt of possibility of polycystic ovary syndrome, estimation and maintenance of menopausal period of elderly female, reduction of risk of osteoporosis and delay of cardiovascular disease. BRIEF DESCRIPTION OF DRAWINGS

[0042] Fig. 1 is a structural schematic diagram of AMH test paper card: 1 is water absorption paper, 2 is nitric acid fiber membrane, 3 is combination pad, 4 is sample pad, 5 is bottom plate, T is detection line film position, and C is quality control line film position.

[0043] Fig. 2 is a schematic diagram of AMH test paper card contained in A type and B type AMH detection box: 6 and 8 are A type card quality control lines, 7 is A type card 1 detection T line (1 ng / mL), 9 is A type card 2 detection T line (10 ng / mL), 10 and 12 are B type card quality control lines, 11 is B type card 1 detection T line (0.5 ng / mL), 13 is B type card 2 detection T line (7.5 ng / mL), and 14 is sample hole. DETAILED DESCRIPTION

[0044] In order to further understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application.

[0045] The experimental methods used in the following embodiments are generally according to conventional conditions or according to the conditions suggested by the manufacturers, unless otherwise specified.

[0046] Figure 1 shows an anti-Mullerian hormone (AMH) test paper card of the present application, comprising a base plate 5, on which a sample pad 4, a conjugate pad 3, a nitrocellulose membrane 2, and an absorbent paper 1 are sequentially attached; one end of the conjugate 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 absorbent paper 1; the nitrocellulose membrane 2 is provided with a test line T and a quality control line C; the test line T is solid-phase immobilized with an AMH monoclonal antibody; and the quality control line C is coated with a goat anti-rabbit IgG. The conjugate pad 3 is sprayed with a latex microsphere complex.

[0047] The anti-Mullerian hormone (AMH) detection kit disclosed in the present application is divided into type A and type B for women of different age groups, each type of detection kit comprising two groups of AMH test paper cards with different sensitivities of detection line, and the conjugate pad of each group of AMH test paper card is sprayed with a latex microsphere complex. The latex microsphere complex comprises a latex microsphere-labeled AMH capture antibody and a rabbit IgG antibody. The AMH test paper cards with different sensitivities of detection line differ in the concentration of the latex microsphere-labeled AMH capture antibody sprayed thereon.

[0048] In the type A AMH detection kit for women aged 18-29, the detection sensitivities of the two groups of AMH test paper cards for AMH are 1 ng / mL and 10 ng / mL, respectively, and the concentrations of the latex microsphere-labeled AMH capture antibody sprayed thereon are 5 μg / mL and 0.5 μg / mL, respectively.

[0049] In the type B AMH detection kit for women aged 30-39, the detection sensitivities of the two groups of AMH test paper cards for AMH are 0.5 ng / mL and 7.5 ng / mL, respectively, and the concentrations of the latex microsphere-labeled AMH capture antibody sprayed thereon are 10 μg / mL and 3.75 μg / mL, respectively.

[0050] The concentration of the latex microsphere-labeled rabbit IgG antibody sprayed on all the AMH test paper cards is 5 μg / mL.

[0051] The above-mentioned latex microsphere-labeled AMH capture antibody and rabbit IgG antibody are both activated by conventional EDC.

[0052] The nucleotide coding sequence of the latex microsphere-labeled AMH capture antibody is shown in SEQ No. 1.

[0053] The nucleotide coding sequence of the AMH monoclonal antibody is shown in SEQ No. 2.

[0054] The rabbit IgG antibody and the goat anti-rabbit IgG polyclonal antibody are commercially available antibodies, both of which are from Xibao Bio. The rabbit IgG antibody has a product number of EXK3226B, and the goat anti-rabbit IgG has a product number of ECY4491A.

[0055] Example 1

[0056] 1. Pretreatment of the bonding pad

[0057] At room temperature, the glass fiber membrane was immersed in 20 mmol Tris buffer (pH 7.4) containing 0.05% Tween-20, 1% bovine serum albumin and 2% trehalose, air-dried for 10 minutes, and then dried at 37°C for 4 hours. After drying, it was sealed and prepared as a binding pad for subsequent gold sputtering.

[0058] 2. Preparation of coating solution

[0059] Anti-AMH monoclonal antibody and goat anti-rabbit IgG antibody were diluted to 1 mg / mL using PB buffer containing 0.9% NaCl + 3% sucrose 10 mM, and then streaked on nitrocellulose membranes at 0.1 μL / mm using a gold streaking spectrometer.

[0060] 3. Sample pad pretreatment

[0061] Immerse the sample pad in 20 mmol of Tris buffer (pH 7.4) containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein, and 500 ng to 5 mg / mL phytohemagglutinin. Air dry at room temperature for 10 minutes, then dry at 37°C for 4 hours. After drying, seal and store for later use.

[0062] 4. Latex microsphere labeling

[0063] (1) Using the conventional EDC method, the latex microspheres were activated in 50mM MES buffer for half an hour;

[0064] (2) Centrifuge at 12000g for 10min and discard the supernatant;

[0065] (3) 20 μL of activated latex microspheres (excess, average particle size 400 nm) were resuspended in 1 mL (50 mM, pH 8.0) of PB buffer, and AMH capture antibody (different for different test strips (see Table 1 for specific amounts) or rabbit IgG antibody (1 μg) was added. After incubation for 1 hour, 20% bovine serum albumin solution was added as blocking solution and reacted for 30 minutes.

[0066] Table 1

[0067] (4) centrifuging the latex microsphere solution obtained in step (3) at 4 DEG C and 12000 rpm for 20 min, removing the supernatant after centrifugation, and re-dissolving the precipitate with a re-dissolving solution, i.e. a PB buffer containing 0.05% Tween 20 and 0.1% BSA, in an amount of 100 uL, to obtain a latex microsphere-labeled AMH capture antibody and a latex microsphere-labeled rabbit IgG antibody, respectively; mixing the two kinds of latex microsphere-labeled antibodies 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 latex microsphere-labeled rabbit IgG antibody is 5 ug / mL (calculated based on the mass concentration of the rabbit IgG antibody).

[0068] (5) spraying the latex microsphere complex obtained in step (4) onto a binding pad at a rate of 0.1 uL / mm using a gold-spraying film-drawing instrument;

[0069] (6) drying the binding pad in an oven at 37 DEG C for 240 min, and sealing and storing the dried binding pad in a desiccator.

[0070] 5. Assembly and plate sticking

[0071] The water absorption paper, the nitrocellulose membrane, the binding pad and the sample pad are sequentially assembled on the bottom plate, and then cut by a high-speed slitting machine to obtain the AMH test strip card product.

[0072] The AMH detection kit is divided into type A and type B for women of different age groups, and each type of detection kit includes two groups of AMH test strip cards with different sensitivities of detection lines. As shown in Figure 2, 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.

[0073] In the present application, the use method of the test strip card is as follows: 1-2 drops (about 15-25 uL) of whole blood sample are slowly added to the sample addition hole of the test strip card, and then 2-3 drops (about 35-45 uL) of sample diluent are added, and the result is read after 15 minutes, and the result may be abnormal after 15 minutes.

[0074] 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), triton 0.01% / L.

[0075] 18-29 years old female is suitable for type A detection kit, and needs to use type A card 1 and type A card 2 respectively for testing. In the case that the quality control line C of type A card 1 and type A card 2 is normally colored; type A card 1 is not detected, it is indicated that the AMH level is lower than 1 ng / mL, and it is prompted that the female has poor ovarian reserve function, and there is a risk of premature ovarian failure; if type A card 2 is detected, it is prompted that the AMH level of the female is too high, and there is a risk of polycystic ovary syndrome; type A card 1 is detected but type A card 2 is not detected, the ovarian reserve function of the female is between 1-10 ng / mL, which belongs to the normal level.

[0076] 30-40 years old female is suitable for type B detection kit, and needs to use type B card 1 and type B card 2 respectively for testing. In the case that the quality control line C of type B card 1 and type B card 2 is normally colored, type B card 1 is not detected, it is indicated that the AMH level is lower than 0.5 ng / mL, and it is prompted that the female has poor ovarian reserve function, and there is a risk of premature ovarian failure; if type B card 2 is detected, it is prompted that the AMH level of the female is too high, and there is a risk of polycystic ovary syndrome; type B card 1 is detected but type B card 2 is not detected, the ovarian reserve function of the female is between 0.5-7.5 ng / mL, which belongs to the normal level.

[0077] When the quality control line of any test paper card does not appear red strip, it may be incorrect operation or reagent has been invalid. At this time, the detection result is invalid, and should be retested.

[0078] The above only describes the preferred examples of the present application and is not used to limit the present application. For the skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made to the present application should be included in the protection scope of the present application.

Claims

1. A latex-based anti-Mullerian hormone test kit, characterized by, The test paper card comprises two groups of test paper cards with different sensitivities, each of which comprises a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper. The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent paper are sequentially connected and pasted on the bottom plate. The conjugate pad is sprayed with a latex microsphere conjugate solution; the latex microsphere conjugate is a latex microsphere-labeled AMH capture antibody and a rabbit IgG antibody. The conjugate pad of the nitrocellulose membrane is sequentially provided with a quality control line and a detection line in parallel from one end of the conjugate pad to one end of the absorbent paper, and the quality control line and the detection line are parallel to the joint seam. The quality control line and the detection line are respectively sprayed with a goat anti-rabbit IgG and an AMH monoclonal antibody. The detection limit of the two groups of test paper cards for anti-Mullerian hormone is optionally one of the following groups: i) 1 ng / mL and 10 ng / mL, ii) 0.5 ng / mL and 7.5 ng / mL.

2. The latex-based anti-Müllerian hormone test kit according to claim 1, characterized in that: The nucleotide coding sequence of the latex microsphere-labeled AMH capture antibody is shown in SEQ No. 1; the nucleotide coding sequence of the AMH monoclonal antibody is shown in SEQ No.

2.

3. The latex-based anti-Müllerian hormone test kit according to claim 1, characterized in that, The two groups of test paper cards with different sensitivities are sprayed with latex microsphere-labeled AMH capture antibodies with different concentrations, and the AMH capture antibody mass concentration is selected from one of the following groups according to the sensitivity requirement: i) 5-25 μg / mL and 0.5-5 μg / mL, preferably 5 μg / mL and 0.5 μg / mL ii) 5-25 μg / mL and 0.5-3.75 μg / mL, preferably 10 μg / mL and 3.75 μg / mL.

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

5. The latex-based anti-Müllerian hormone test kit according to claim 1, wherein, The average particle size of the latex microsphere particles is 100-600 nm, preferably 400 nm.

6. The latex-based anti-Müllerian hormone test kit according to claim 1, wherein, The concentration of the AMH monoclonal antibody and the goat anti-rabbit IgG is 1 mg / mL, the spraying amount is 0.8 uL / cm, the antibodies are sprayed on the nitrocellulose membrane respectively to form the detection line and the quality control line, the interval between the two lines is 5 mm, and after drying, it is sealed for standby.

7. The latex-based anti-Müllerian hormone test kit according to claim 1, characterized in that, The conjugate pad is selected from a glass fiber membrane; the pretreatment method is to immerse the glass fiber membrane in a 20 mmol Tris buffer solution containing 0.05% Tween-20, 1% bovine serum albumin and 2% trehalose, dry at room temperature for 10 minutes, and then dry at 37°C for 4 hours.

8. The latex-based anti-Müllerian hormone test kit according to claim 1, wherein, The pretreatment method of the sample pad is to immerse the sample pad in a 20 mmol Tris buffer solution containing 0.05% Tween-20, 1% bovine serum albumin, 0.3% casein and 500 ng-5 mg / mL phytohemagglutinin, dry at room temperature for 10 minutes, and then dry at 37°C for 4 hours.

9. The latex-based anti-Müllerian hormone test kit according to claim 1, wherein, It also includes a sample diluent; the sample diluent is an aqueous solution of a mixture of sodium chloride, dodecahydrate disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride and triton.

10. The latex agglutination anti-Mullerian hormone test kit according to claim 9, characterized by, The concentrations of the substances are respectively: Sodium chloride 8 g / L; Dodecahydrate disodium hydrogen phosphate 0.126 g / L; Potassium dihydrogen phosphate 0.06 g / L; Potassium chloride 0.4 g / L; Triton X-100 0.01 % / L.

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