Antibodies against AMH, reagents and kits for detecting AMH

Abstract

The application discloses an antibody against AMH, a reagent and a kit for detecting AMH, and relates to the technical field of antibodies.The antibody against AMH disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region.The antibody has good affinity to AMH, and the use of the antibody for detecting AMH has better sensitivity and specificity.The application provides more antibody options for the detection of AMH, and the performance of the antibody is better.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an anti-AMH antibody, a reagent for detecting AMH, and a kit. Background Technology

[0002] Anti-Mullerian hormone (AMH) is a member of the transforming growth factor β superfamily, first discovered by Professor Alfred Jost in 1974. It is a diglycoprotein composed of two 70kD protein subunits linked by disulfide bonds, with a relative molecular mass of 140kD. Its main function is to influence follicle growth and differentiation. The human gene encoding AMH is located on chromosome 19p13.3, is 2.4–2.8kb in size, and contains 5 exons. Studies have shown that the N-terminal region of AMH plays a crucial role in maintaining the protein's integrity and activity. During metabolic transport, a specific site (amino acid 451) of AMH can be cleaved to form a precursor fragment (pro-AMH, 26–451 aar) and a mature fragment (Mature AMH, 452–560 aar), and the two cleaved fragments can then be linked together non-covalently.

[0003] Alpha-methylhydrochloride (AMH) plays a crucial role in the development of gonadal organs and is one of the important markers of gonadal function in both men and women. In Sertoli cells of male fetuses, the transcription factor SOX-9 (SOX9) activates AMH to bind to Anti-Mullerian Hormone Receptor Type II (AMHR2), leading to the regression of Müllerian ducts in male embryos. This prevents the formation of fallopian tubes, uterus, and upper vagina, allowing for normal development of the male reproductive tract. In female embryos, the Müllerian ducts differentiate into the uterine canal and fallopian tubes. AMH is also a product of the granulosa cells of preantral and small antral follicles in women. From puberty onwards, serum AMH levels gradually decrease over time, reaching levels undetectable by ELISA during menopause. The normal range for AMH is 2-6.8 ng / ml. Higher values ​​indicate a greater abundance of oocytes and a longer optimal period for conception, while lower values ​​indicate poorer ovarian function. After age 35, AMH levels begin to decline sharply. When levels fall below 0.7 ng / ml, it indicates a severely depleted egg reserve, making conception nearly impossible. A level above 6.8 may suggest a predisposition to polycystic ovary syndrome (PCOS). The use of ovulation-inducing injections or medications can also cause the ovaries to overreact and release too many eggs, leading to ovarian hyperstimulation syndrome (OHSS). Therefore, AMH levels can be used to assess egg reserves and ovarian function, and can serve as a diagnostic criterion for ovarian responsiveness, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS) in in vitro fertilization (IVF) combined with embryo transfer.

[0004] Currently, the commonly used methods for detecting AMH in clinical practice both domestically and internationally include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECIA), and chemiluminescence immunoassay. ELISA is time-consuming, has low sensitivity, high background levels, and is prone to false positives and false negatives, and requires professional operation. ECA and chemiluminescence immunoassays, such as Roche's ELECSYS AMH analyzer, are gradually replacing ELISA, but they are expensive and unsuitable for single-person or small-batch testing. Furthermore, they require specialized personnel for operation, resulting in high maintenance and testing costs, making widespread adoption impractical. All of these methods require the use of anti-AMH monoclonal antibodies, but currently, there are few AMH monoclonal antibody products available, their performance varies, and their specificity and sensitivity are also deficient.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-AMH antibody, a reagent for detecting AMH, and a kit. The antibody provided by this invention can specifically bind to AMH, has good affinity for AMH, and the detection of AMH using this antibody has high sensitivity and specificity.

[0007] This invention is implemented as follows:

[0008] On one hand, the present invention provides an anti-AMH antibody or a functional fragment thereof, said antibody or functional fragment having the following complementarity-determining regions:

[0009] CDR-VH1: GF-X1-FS-X2-FGMS; where: X1 is S or T; X2 is I, V or L;

[0010] CDR-VH2: T-X1-SNGG-X2-YTYYP-X3-S-X4-KG; where: X1 is L or I; X2 is T or S; X3 is E or D; X4 is I, V or L;

[0011] CDR-VH3: X1-RHPR-X2-NG-X3-DGAM; where: X1 is A or T; X2 is I, V or L; X3 is Y, F or S;

[0012] CDR-VL1: AS-X1-S-X2-DNYD-X3-SFM; where: X1 is Q or E; X2 is I, V or L; X3 is I or L;

[0013] CDR-VL2: AASN-X1-X2-S; where: X1 is K, Q, or R; X2 is G or A;

[0014] CDR-VL3: QQS-X1-E-X2-PW; where: X1 is K and R; X2 is L, V or I.

[0015] The antibody or functional fragment thereof targeting AMH provided by this invention has the aforementioned complementarity-determining region structure; the antibody or functional fragment thereof can specifically bind to AMH and has good affinity for AMH. Using this antibody to detect AMH provides high sensitivity and specificity. This invention provides a wider range of antibody options with better performance for AMH.

[0016] In an optional implementation, X1 is I in CDR-VH2; X1 is T in CDR-VH3; X3 is I in CDR-VL1; X2 is G in CDR-VL2; and X1 is K in CDR-VL3.

[0017] Embodiments of the present invention have shown that when the mutation site in the complementarity-determining region is an amino acid residue, the antibody exhibits better affinity for AMH.

[0018] In an optional implementation, in CDR-VH1, X1 is S.

[0019] In an optional implementation, in CDR-VH1, X1 is T.

[0020] In an optional implementation, X2 is I in CDR-VH1.

[0021] In an optional implementation, in CDR-VH1, X2 is V.

[0022] In an optional implementation, in CDR-VH1, X2 is L.

[0023] In an optional implementation, X2 in CDR-VH2 is T.

[0024] In an optional implementation, X2 in CDR-VH2 is S.

[0025] In an optional implementation, in CDR-VH2, X3 is E.

[0026] In an optional implementation, in CDR-VH2, X3 is D.

[0027] In an optional implementation, in CDR-VH2, X4 is I.

[0028] In an optional implementation, in CDR-VH2, X4 is V.

[0029] In an optional implementation, in CDR-VH2, X4 is L.

[0030] In an optional implementation, in CDR-VH3, X2 is I.

[0031] In an optional implementation, in CDR-VH3, X2 is V.

[0032] In an optional implementation, in CDR-VH3, X2 is L.

[0033] In an optional implementation, X3 in CDR-VH3 is Y.

[0034] In an optional implementation, X3 in CDR-VH3 is F.

[0035] In an optional implementation, X3 in CDR-VH3 is S.

[0036] In an optional implementation, X1 in CDR-VL1 is Q.

[0037] In an optional implementation, in CDR-VL1, X1 is E.

[0038] In an optional implementation, in CDR-VL1, X2 is I.

[0039] In an optional implementation, in CDR-VL1, X2 is V.

[0040] In an optional implementation, in CDR-VL1, X2 is L.

[0041] In an optional implementation, X1 is K in CDR-VL2.

[0042] In an optional implementation, X1 in CDR-VL2 is Q.

[0043] In an optional implementation, in CDR-VL2, X1 is R.

[0044] In an optional implementation, in CDR-VL3, X2 is L.

[0045] In an optional implementation, in CDR-VL3, X2 is V.

[0046] In an optional implementation, in CDR-VL3, X2 is I.

[0047] In an optional embodiment, the complementarity-determining regions of the antibody or its functional fragment are selected from any one of the following mutation combinations 1-55:

[0048]

[0049]

[0050] In an optional embodiment, the antibody or its functional fragment is combined with AMH at K D ≤2.6×10 -8 Affinity binding at mol / L.

[0051] In an optional implementation, K D ≤2×10 -8 mol / L, K D ≤1×10 -8 mol / L, K D ≤9×10 -9 mol / L, K D ≤8×10 -9 mol / L, K D ≤7×10 -9 mol / L, KD ≤6×10 -9 mol / L, K D ≤5×10 -9 mol / L, K D ≤4×10 -9 mol / L, K D ≤3×10 -9 mol / L, K D ≤2×10 -9 mol / L or K D ≤1×10 -9 mol / L.

[0052] In an optional implementation, K D ≤9×10 -9 mol / L.

[0053] In an optional implementation, 1.52 × 10 -9 mol / L≤K D ≤8.94×10 -9 mol / L.

[0054] K D The detection is performed according to the method in the embodiments of the present invention.

[0055] In an optional implementation, X1 is L in CDR-VH2; X1 is A in CDR-VH3; X3 is L in CDR-VL1; X2 is A in CDR-VL2; and X1 is R in CDR-VL3.

[0056] In an optional embodiment, the complementarity-determining regions of the antibody or its functional fragment are selected from any one of the following mutation combinations 56-62:

[0057]

[0058] In an optional embodiment, the antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:1-4, and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:5-8.

[0059] Typically, the variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0060] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8) mentioned above.

[0061] In an optional implementation, the antibody further includes a constant region.

[0062] In an optional implementation, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0063] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0064] In an optional implementation, the constant region is derived from mice.

[0065] In an optional embodiment, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.

[0066] In an optional embodiment, the functional fragment is selected from any one of the antibody’s VHH, F(ab')2, Fab', Fab, Fv and scFv.

[0067] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0068] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0069] On the other hand, the present invention provides a reagent or kit for detecting AMH, comprising an antibody or a functional fragment thereof as described in any of the preceding claims.

[0070] In an optional embodiment, the antibody or its functional fragment in the above reagent or kit is labeled with a detectable marker.

[0071] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0072] In optional embodiments, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0073] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0074] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0075] In optional embodiments, the enzymes that catalyze the color development of the substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0076] In optional embodiments, the radioactive isotope includes, but is not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0077] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0078] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0079] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0080] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0081] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its functional fragment.

[0082] On the other hand, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0083] On the other hand, the present invention provides recombinant cells containing the above-described carrier.

[0084] On the other hand, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing recombinant cells as described above, and separating and purifying the antibody or the functional fragment thereof from the culture product.

[0085] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in this invention, those skilled in the art will readily conceive of preparing the antibody or its functional fragment using genetic engineering or other techniques (chemical synthesis, hybridoma cells), such as isolating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention. Attached Figure Description

[0086] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0087] Figure 1 The results of reducing SDS-PAGE of the anti-AMH antibody in Example 1 are shown. Detailed Implementation

[0088] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0090] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.C. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0091] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0092] Example 1

[0093] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen.

[0094] 1. Construction of recombinant plasmids

[0095] (1) Antibody gene preparation

[0096] mRNA was extracted from hybridoma cell lines secreting anti-AMH antibodies, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0097] (2) Sequence analysis of antibody variable region genes

[0098] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 336 bp, belonging to the VkII gene family, and it had a 57 bp leader peptide sequence in front of it. Among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 366 bp, belonging to the VH1 gene family, and it had a 57 bp leader peptide sequence in front of it.

[0099] (3) Construction of recombinant antibody expression plasmid

[0100] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.74kb Light Chain gene fragment and the 1.46kb Heavy Chain gene fragment were amplified by PCR.

[0101] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0102] 2. Screening of stable cell lines

[0103] (1) Transient transfection of CHO cells with recombinant antibody expression plasmid to determine plasmid activity.

[0104] The plasmid was diluted to 400 ng / ml with ultrapure water and used to regulate CHO cells to 1.43 × 10⁻⁶. 7 Cells / ml were collected in centrifuge tubes, 100 μL of plasmid was mixed with 700 μL of cells, transferred to an electroporation cuvette, and electroporated. Samples were taken and counted on days 3, 5, and 7, and samples were collected and analyzed on day 7.

[0105] Dilute AMH antigen to 1 μg / ml with coating buffer, 100 μL per well, and incubate overnight at 4°C. The next day, wash twice with washing buffer and blot dry. Add blocking buffer (20% BSA + 80% PBS), 120 μL per well, incubate at 37°C for 1 h, and blot dry. Add diluted cell supernatant, 100 μL / well, incubate at 37°C for 30 min. Wash five times with washing buffer and blot dry. Add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37°C for 3 h. 0 min; wash 5 times with washing buffer and pat dry; add chromogenic solution A (50 μL / well, containing citric acid + sodium acetate + acetanilide + urea peroxide), add chromogenic solution B (50 μL / well, containing citric acid + EDTA·2Na + TMB + concentrated HCl), 10 min; add stop solution (50 μL / well, containing EDTA·2Na + concentrated H2SO4); read the OD value at 450 nm (reference 630 nm) on the microplate reader. The results showed that the OD of the reaction was still greater than 1.0 after 1000-fold dilution of cell supernatant, and the OD of the reaction in wells without cell supernatant was less than 0.1, indicating that the antibody generated after transient plasmid transfection is active against AMH antigen.

[0106] (2) Linearization of recombinant antibody expression plasmid

[0107] Prepare the following reagents: Buffer 50 μL, DNA 100 μg / tube, PuvI enzyme 10 μL, and sterile water to a final volume of 500 μL. Incubate overnight at 37°C. Extract with an equal volume of phenol / chloroform / isoamyl alcohol (lower layer) in a 25:24:1 ratio, followed by extraction with chloroform (aqueous phase). Precipitate on ice with 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volume of ethanol. Rinse the precipitate with 70% ethanol to remove organic solvents. After the ethanol has completely evaporated, reconstitute with an appropriate amount of sterile water. Finally, determine the concentration.

[0108] (3) Stable transfection with recombinant antibody expression plasmid, followed by pressure screening to select stable cell lines.

[0109] The plasmid was diluted to 400 ng / ml with ultrapure water and used to regulate CHO cells to 1.43 × 10⁻⁶. 7 Cells / ml were collected in centrifuge tubes, 100 μL of plasmid was mixed with 700 μL of cells, transferred to an electroporation cuvette, electroporated, and counted the cells the next day; then cultured in 25 μmol / L MSX 96-well pressurized culture for about 25 days.

[0110] Observe the labeled clone wells containing cells under a microscope and record the degree of confluence; collect the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration to transfect 24-well cells, and transfect 6-well cells after about 3 days; after 3 days, preserve the culture in batch culture and adjust the cell density to 0.5 × 10⁻⁶ cells / well. 6 Cells / ml, 2.2ml batch culture, cell density 0.3×10⁶ 6 Cells / ml, 2ml for seeding; 6-well batch culture supernatant sent for testing after 7 days, cell lines with smaller antibody concentration and cell diameter were selected for TPP seeding and passage.

[0111] 3. Recombinant Antibody Production

[0112] (1) Cell expansion

[0113] After cell resuscitation, cells were initially cultured in 125ml shake flasks with an inoculation volume of 30ml in 100% Dynamis medium. The flasks were placed in a shaker at 120 rpm, 37°C, and 8% CO2. After 72 hours of culture, cells were propagated at a density of 500,000 cells / ml. The propagation volume was calculated based on production requirements, and the medium was 100% Dynamis. Propagation was then repeated every 72 hours. Once the cell count met production needs, the inoculation density was strictly controlled at approximately 500,000 cells / ml for production.

[0114] (2) Shake flask production and purification

[0115] Shake flask parameters: rotation speed 120 rpm, temperature 37℃, carbon dioxide 8%. Feeding: Feeding began daily after 72 hours of incubation in the shake flasks. HyClone™ Cell Boost™ Feed 7a was used to feed 3% of the initial culture volume daily, and Feed 7b was used to feed 1 / 1000 of the initial culture volume daily, continuing until day 12 (feeding on day 12). Glucose was added at 3 g / L on day 6. Samples were collected on day 13. Affinity purification was performed using a protein A affinity chromatography column. 4 μg of purified antibody was subjected to reducing SDS-PAGE, with 4 μg of exogenous control antibody used as a control. The electrophoresis results are shown below. Figure 1 As shown, two bands are displayed after reducing SDS-PAGE: one with Mr 50 KD (heavy chain, SEQ ID NO:14) and the other with Mr 28 KD (light chain, SEQ ID NO:13).

[0116] Example 2

[0117] Antibody performance testing

[0118] (1) Example 1 Activity detection of antibodies and their mutants

[0119] The antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region is shown in SEQ ID NO:12. The amino acid sequences of the complementarity-determining regions on the heavy chain variable region are as follows:

[0120] CDR-VH1: GFT(X1)-FSV(X2)-FGMS;

[0121] CDR-VH2: TL(X1)-SNGS(X2)-YTYYPD(X3)-SL(X4)-KG;

[0122] CDR-VH3: A(X1)-RHPRV(X2)-NGY(X3)-DGAM;

[0123] Its light chain variable region is shown in SEQ ID NO:11, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0124] CDR1-VL: ASE(X1)-SI(X2)-DNYDL(X3)-SFM;

[0125] CDR-VL2: AASNK(X1)-A(X2)-S;

[0126] CDR-VL3: QQSR(X1)-EI(X2)-PW.

[0127] Based on the anti-AMH antibody (WT) of Example 1, mutations were made at sites related to antibody activity in the complementarity-determining region, where X1, X2, X3, and X4 are mutation sites. See Table 1 below.

[0128] Table 1 Mutation sites related to antibody activity

[0129]

[0130] Assay for antibody binding activity in Table 1:

[0131] Coating buffer (mainly NaHCO3) was used to dilute AMH antigen to 1 μg / ml for coating microplates, 100 μl per well, incubated overnight at 4°C. The next day, the plates were washed twice with washing buffer and patted dry. Blocking buffer (20% BSA + 80% PBS) was added, 120 μl per well, incubated at 37°C for 1 hour, and patted dry. Diluted AMH monoclonal antibody was added, 100 μl / well, incubated at 37°C for 30-60 minutes. The plates were washed five times with washing buffer and patted dry. Goat... Anti-mouse IgG-HRP, 100 μl per well, incubated at 37℃ for 30 min; wash 5 times with washing buffer (PBS), pat dry; add chromogenic solution A (50 μl / well, containing 2.1 g / L citric acid, 12.25 g / L citric acid, 0.07 g / L acetanilide, and 0.5 g / L urea peroxide), add chromogenic solution B (50 μl / well, containing 1.05 g / L citric acid, 0.186 g / L EDTA·2Na, 0.45 g / L TMB, and 0.2 ml / L concentrated HCl), incubate for 10 min; add stop solution (50 μl / well, containing 0.75 g / L EDTA·2Na and 10.2 ml / L concentrated H2SO4); read the OD value at 450 nm (reference 630 nm) on a microplate reader. The results are shown in Table 2 below.

[0132] Table 2 Activity data of WT antibodies and their mutants

[0133] Antibody concentration (ng / ml) 250 125 62.5 31.25 15.625 0 WT 1.952 1.621 1.182 0.650 0.360 0.046 Mutation 1 2.411 2.382 2.221 1.811 1.016 0.082 Mutation 2 2.45 2.383 2.167 1.503 0.856 0.05 Mutation 3 2.312 2.323 2.107 1.616 0.926 0.063 Mutation 4 2.396 2.046 1.882 0.955 0.860 0.146 Mutation 5 0.653 0.105 - - - - Mutation 6 0.426 0.063 - - - -

[0134] Table 2 shows that WT, mutations 1 to 4 have better activity than mutations 5 and 6, with mutation 1 showing the best activity.

[0135] (2) Affinity detection of antibodies and their mutants

[0136] (a) Based on mutation 1, other sites were mutated. The sequences of each mutation are shown in Table 3 below.

[0137] Table 3 Mutation sites related to antibody affinity

[0138]

[0139]

[0140] Affinity Analysis

[0141] Using the AMC sensor, the purified antibody was diluted to 10 ug / mL with PBST, and the AMH antigen was serially diluted with PBST to 45 ug / mL, 22.5 ug / mL, 11.3 ug / mL, 5.60 ug / mL, 2.80 ug / mL, 1.40 ug / mL, and 0.70 ug / mL.

[0142] Operating procedure: Equilibrate in Buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in Buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in Buffer 2 for 1200s, regenerate the sensor using 10mM pH 1.69 GLY solution and Buffer 3, and output data. K D The constant represents the equilibrium dissociation constant, i.e., affinity; kon represents the binding rate; and kdis represents the dissociation rate. The results are shown in Table 4 below.

[0143] Table 4 Affinity Test Data

[0144]

[0145]

[0146] The data in Table 4 show that Mutation 1 and its series of mutants all have good affinity, indicating that the antibodies obtained by mutation according to the mutation methods in Table 3 based on Mutation 1 all have good affinity.

[0147] (b) Based on WT, other sites were mutated and the affinity of each mutant was tested. The sequences of each mutation are shown in Table 5 below, and the corresponding affinity data are shown in Table 6.

[0148] Table 5 Mutations performed using WT as the backbone

[0149]

[0150] Table 6 Affinity test results of WT antibodies and their mutants

[0151] <![CDATA[K D (M)]]> kon(1 / Ms) kdis(1 / s) WT 2.57E-08 1.01E+04 2.60E-04 WT 1 1.56E-08 2.43E+04 3.78E-04 WT 2 2.08E-08 1.82E+04 3.79E-04 WT 3 1.70E-08 2.63E+04 4.48E-04 WT 4 1.51E-08 2.65E+04 4.01E-04 WT5 1.30E-08 2.69E+04 3.50E-04 WT6 1.78E-08 2.19E+04 3.89E-04

[0152] The data in Table 6 show that WT and its series of mutants all have good affinity, indicating that the antibodies obtained by mutation according to the mutation methods in Table 5 based on WT all have good affinity.

[0153] (3) Stability assessment of bare resistors

[0154] The above-mentioned antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the above-mentioned antibodies were stable. Table 7 below shows the OD results of enzyme immunoassay for mutant 1 antibody after 21 days of testing.

[0155] Table 7

[0156] Sample concentration (ng / ml) 100 10 0 4℃, 21-day sample 1.985 0.528 0.05 -80℃, 21-day sample 1.93 0.538 0.045 37℃, 21-day sample 1.931 0.593 0.051

[0157] (4) Application performance testing

[0158] Using the antibodies listed in Table 3 in combination with another AMH antibody, a double-antibody sandwich method was employed. The specificity reached 100% and the sensitivity was between 99% and 99.8% on the chemiluminescence platform.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Dongguan Pengzhi Biotechnology Co., Ltd. <120> Anti-AMH antibodies, reagents and kits for detecting AMH <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> PRT <213> Artificial sequence <400> 1 Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg 20 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <400> 2 Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 3 <211> 32 <212> PRT <213> Artificial sequence <400> 3 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 1 5 10 15 Leu Asn Ile His Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys 20 25 30 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <400> 4 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <210> 5 <211> 25 <212> PRT <213> Artificial sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 25 <210> 6 <211> 14 <212> PRT <213> Artificial sequence <400> 6 Trp Phe Arg Gln Ser Pro Asp Lys Arg Leu Glu Trp Val Ala 1 5 10 <210> 7 <211> 30 <212> PRT <213> artificial sequence <400> 7 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Ala Ser Leu Arg Ser Val Asp Thr Ala Met Tyr Tyr Cys 20 25 30 <210> 8 <211> 13 <212> PRT <213> artificial sequence <400> 8 Asp Tyr Trp Gly Gln Gly Ile Ser Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 106 <212> PRT <213> artificial sequence <400> 9 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 1 5 10 15 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 20 25 30 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 35 40 45 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 65 70 75 80 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 85 90 95 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 10 <211> 336 <212> PRT <213> Artificial Sequence <400> 10 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val 65 70 75 80 Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys 85 90 95 Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys 100 105 110 Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser 115 120 125 Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu 130 135 140 Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 145 150 155 160 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 165 170 175 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val 180 185 190 Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 195 200 205 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr 210 215 220 Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu 225 230 235 240 Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys 245 250 255 Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser 260 265 270 Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp 275 280 285 Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser 290 295 300 Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly 305 310 315 320 Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys 325 330 335 <210> 11 <211> 112 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Ile Asp Asn Tyr 20 25 30 Asp Leu Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Lys Ala Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 <210> 12 <211> 122 <212> PRT <213> Artificial Sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Phe 20 25 30 Gly Met Ser Trp Phe Arg Gln Ser Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Leu Ser Asn Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ala Ser Leu Arg Ser Val Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Pro Arg Val Asn Gly Tyr Asp Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Ile Ser Val Thr Val Ser Ser 115 120 <210> 13 <211> 218 <212> PRT <213> Synthetic Sequence <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Ile Asp Asn Tyr 20 25 30 Asp Leu Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Lys Ala Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 14 <211> 458 <212> PRT <213> Synthetic Sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Phe 20 25 30 Gly Met Ser Trp Phe Arg Gln Ser Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Leu Ser Asn Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ala Ser Leu Arg Ser Val Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Pro Arg Val Asn Gly Tyr Asp Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Ile Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro 165 170 175 Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His 195 200 205 Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro 210 215 220 Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys 225 230 235 240 Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 245 250 255 Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys 260 265 270 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 275 280 285 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 290 295 300 Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln 305 310 315 320 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 325 330 335 Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly 340 345 350 Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln 355 360 365 Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn 370 375 380 Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu 385 390 395 400 Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 405 410 415 Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp 420 425 430 Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu 435 440 445 Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys 450 455

Claims

1. An antibody against AMH or a functional fragment thereof, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VH1: GF-X1-FS-X2-FGMS; CDR-VH2: T-X1-SNGG-X2-YTYYP-X3-S-X4-KG; where: X1 is I; CDR-VH3: X1-RHPR-X2-NG-X3-DGAM; where: X1 is T; CDR-VL1: AS-X1-S-X2-DNYD-X3-SFM; where: X3 is I; CDR-VL2: AASN-X1-X2-S; where: X2 is G; CDR-VL3: QQS-X1-E-X2-PW; where: X1 is K; Each complementarity-determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 1-55: 。 2. An anti-AMH antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VH1: GF-X1-FS-X2-FGMS; CDR-VH2: T-X1-SNGG-X2-YTYYP-X3-S-X4-KG; where: X1 is L; CDR-VH3: X1-RHPR-X2-NG-X3-DGAM; where: X1 is A; CDR-VL1: AS-X1-S-X2-DNYD-X3-SFM; where: X3 is L; CDR-VL2: AASN-X1-X2-S; where: X2 is A; CDR-VL3: QQS-X1-E-X2-PW; where: X1 is R; Each complementarity-determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 56-62: 。 3. The antibody or its functional fragment according to claim 1, characterized in that, The antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:1-4, and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:5-8.

4. The antibody or its functional fragment according to claim 2, characterized in that, The antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:1-4, and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:5-8.

5. The anti-AMH antibody or its functional fragment according to any one of claims 1-4, characterized in that, The antibody also contains a constant region.

6. The anti-AMH antibody or its functional fragment according to claim 5, characterized in that, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

7. The anti-AMH antibody or its functional fragment according to claim 5, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

8. The anti-AMH antibody or its functional fragment according to claim 7, characterized in that, The species source of the constant region is dairy cows.

9. The anti-AMH antibody or its functional fragment according to claim 7, characterized in that, The species source of the constant region is turkey or fighting cock.

10. The anti-AMH antibody or its functional fragment according to claim 7, characterized in that, The constant region was derived from mice.

11. The anti-AMH antibody or its functional fragment according to claim 10, characterized in that, The light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:

10.

12. The anti-AMH antibody or its functional fragment according to any one of claims 1-4, characterized in that, The functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

13. A reagent or kit for detecting AMH, characterized in that, It includes the antibody or its functional fragment as described in any one of claims 1-12.

14. The reagent or kit according to claim 13, characterized in that, The antibody or its functional fragment is labeled with a detectable marker.

15. The reagent or kit according to claim 14, characterized in that, The detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

16. The reagent or kit according to claim 15, characterized in that, The fluorescent dyes are selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives.

17. The reagent or kit according to claim 15, characterized in that, The enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

18. The reagent or kit according to claim 15, characterized in that, The radioactive isotope is selected from 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

19. The reagent or kit according to claim 15, characterized in that, The chemiluminescent reagent is selected from luminol and its derivatives, luciferin, fluorescein from crustaceans and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

20. The reagent or kit according to claim 15, characterized in that, The nanoparticle markers are selected from nanoparticles and colloids.

21. The reagent or kit according to claim 20, characterized in that, The nanoparticles are selected from at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

22. The reagent or kit according to claim 20, characterized in that, The colloid is selected from latex, colloidal selenium, colloidal metal, disperse dyes, and dye-labeled microspheres.

23. The reagent or kit according to claim 22, characterized in that, The colloidal metal is selected from colloidal gold and colloidal silver.

24. A carrier, characterized in that, It contains a nucleic acid molecule encoding an antibody or a functional fragment thereof as described in any one of claims 1-12.

25. A recombinant cell, characterized in that, It contains the carrier as described in claim 24.

26. A method for preparing an antibody or a functional fragment thereof as described in any one of claims 1-12, characterized in that, It includes: The recombinant cells of claim 25 are cultured, and the antibody or its functional fragment is isolated and purified from the culture product.

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