CN114478764A

CN114478764A - anti-AMH antibody, reagent and kit for detecting AMH

Info

Publication number: CN114478764A
Application number: CN202011259343.1A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 叶庆妮; 陈晓倩; 王晨; 范凌云
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2022-05-13
Also published as: WO2022100263A1

Abstract

The invention discloses an anti-AMH antibody, a reagent and a kit for detecting AMH, and relates to the technical field of antibodies. The anti-AMH antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has better affinity to AMH, and the detection of AMH by using the antibody has better sensitivity and specificity. The invention provides more antibody selection with better performance for AMH detection.

Description

anti-AMH antibody, reagent and kit for detecting AMH

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-AMH antibody, a reagent for detecting AMH and a kit.

Background

Anti-Mullerian Hormone (Anti-Mullerian Hormone, AMH) is a member of the transforming growth factor beta superfamily, first discovered by Professor Alfred Jost in 1974. It is a dimeric glycoprotein composed of two 70kD protein subunits through disulfide bonds, with a relative molecular mass of 140kD, and its main role is to influence the growth and differentiation of follicles. The human gene for coding AMH is located on chromosome 19p13.3, has the size of 2.4-2.8 kb and contains 5 exons. It was shown that the N-terminal region of AMH plays a key role in maintaining the intact activity of the protein, that during metabolic transport, a specific site (amino acid 451) of AMH can be cleaved to form a precursor fragment (pro-AMH, 26-451aar) and a Mature fragment (Mature AMH, 452 and 560aar), and that the two cleaved fragments can be linked together again by non-covalent bonds.

AMH plays an important role in the development process of gonadal organs and is one of important markers of gonadal functions of men and women. In Sertoli cells of male fetuses, the Transcription factor SOX-9 (SOX 9) activates AMH to bind to Anti-Mullerian Hormone Receptor Type II (Anti-Mullerian Hormone Receptor Type II, AMHR2) and causes the degeneration of Mullerian tubes in male embryos, thereby preventing the formation of the oviducts, uterus and upper vagina, resulting in the normal development of the male reproductive tract. Whereas in female embryos the muller's canal will differentiate into the uterine canal and the fallopian tube. AMH is also a product of pre-antral and antral follicular granulosa cells in women, and from puberty, serum levels of AMH slowly decrease over time and in menopause to levels undetectable by ELISA. The normal value of AMH is between 2 ng/ml and 6.8ng/ml, the higher the value is, the more abundant the ovum stock is, the longer the golden period suitable for conception, and the lower the value is, the worse the ovary function is. After age 35, the AMH value begins to decrease sharply, and when the AMH value is lower than 0.7ng/ml, the egg stock is seriously insufficient and is hardly pregnant. If the value is greater than 6.8, the constitution with polycystic ovarian syndrome is considered. When ovulatory injections or drugs are used, ovaries are easy to excessively react and discharge excessive ova, so that ovarian hyperstimulation syndrome is caused. Therefore, the ovum stock and the ovary function can be judged by detecting the AMH level, and the method can be used as the diagnosis basis of the ovary reactivity, polycystic ovary syndrome (PCOS) and ovarian hyperstimulation syndrome (OHSS) of in vitro fertilization combined embryo transfer (IVF).

At present, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence and chemiluminescence methods are commonly used for clinically detecting AMH at home and abroad. The ELISA method is long in time consumption, low in sensitivity, high in background value, prone to cause false positive and false negative results, and needs to be operated by professional personnel. The ELISA method is gradually replaced by the electrochemiluminescence method and the chemiluminescence method such as the Roche ELECSYS AMH detector at present, but the electrochemiluminescence method and the chemiluminescence method are expensive, are not suitable for single-person detection and small-batch detection, and are not suitable for large-scale popularization and use due to the fact that special instrument operators are needed and the maintenance and detection costs are high. The methods all need to use monoclonal antibodies against AMH, but the conventional AMH monoclonal antibodies have fewer products, different performances and defects of specificity and sensitivity.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an antibody against AMH, a reagent and a kit for detecting AMH. The antibody provided by the invention can be specifically combined with AMH, has better affinity to AMH, and has higher sensitivity and specificity when being used for detecting AMH.

The invention is realized by the following steps:

in one aspect, the invention provides an anti-AMH antibody or functional fragment thereof, said antibody or functional fragment thereof having the complementarity determining regions:

CDR-VH 1: G-F-X1-F-S-X2-F-G-M-S; wherein: x1 is S or T; x2 is I, V or L;

CDR-VH 2: T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G; wherein: x1 is L or I; x2 is T or S; x3 is E or D; x4 is I, V or L;

CDR-VH 3: X1-R-H-P-R-X2-N-G-X3-D-G-A-M; wherein: x1 is A or T; x2 is I, V or L; x3 is Y, F or S;

CDR-VL 1: A-S-X1-S-X2-D-N-Y-D-X3-S-F-M; wherein: x1 is Q or E; x2 is I, V or L; x3 is I or L;

CDR-VL 2: A-A-S-N-X1-X2-S; wherein: x1 is K, Q or R; x2 is G or A;

CDR-VL 3: Q-Q-S-X1-E-X2-P-W; wherein: x1 is K and R; x2 is L, V or I.

The antibody or functional fragment thereof against AMH provided by the invention has the complementarity determining region structure; the antibody or the functional fragment thereof can be specifically combined with AMH, has better affinity to AMH, and has higher sensitivity and specificity when being used for detecting AMH. The invention provides more and better antibody selection for AMH.

In alternative embodiments, in CDR-VH2, X1 is I; in CDR-VH3, X1 is T; in CDR-VL1, X3 is I; in CDR-VL2, X2 is G; in CDR-VL3, X1 is K.

The examples of the present invention have found that when the mutation site in the complementarity determining region is the amino acid residue, the antibody exhibits a better affinity for AMH.

In an alternative embodiment, in CDR-VH1, X1 is S.

In an alternative embodiment, in CDR-VH1, X1 is T.

In an alternative embodiment, in CDR-VH1, X2 is I.

In an alternative embodiment, in CDR-VH1, X2 is V.

In an alternative embodiment, in CDR-VH1, X2 is L.

In an alternative embodiment, in CDR-VH2, X2 is T.

In an alternative embodiment, in CDR-VH2, X2 is S.

In an alternative embodiment, in the CDR-VH2, X3 is E.

In an alternative embodiment, in CDR-VH2, X3 is D.

In an alternative embodiment, in CDR-VH2, X4 is I.

In an alternative embodiment, in the CDR-VH2, X4 is V.

In an alternative embodiment, in CDR-VH2, X4 is L.

In an alternative embodiment, in CDR-VH3, X2 is I.

In an alternative embodiment, in CDR-VH3, X2 is V.

In an alternative embodiment, in CDR-VH3, X2 is L.

In an alternative embodiment, in CDR-VH3, X3 is Y.

In an alternative embodiment, in CDR-VH3, X3 is F.

In an alternative embodiment, in the CDR-VH3, X3 is S.

In an alternative embodiment, in CDR-VL1, X1 is Q.

In an alternative embodiment, in CDR-VL1, X1 is E.

In an alternative embodiment, in CDR-VL1, X2 is I.

In an alternative embodiment, in CDR-VL1, X2 is V.

In an alternative embodiment, in CDR-VL1, X2 is L.

In an alternative embodiment, in CDR-VL2, X1 is K.

In an alternative embodiment, in CDR-VL2, X1 is Q.

In an alternative embodiment, in CDR-VL2, X1 is R.

In an alternative embodiment, in CDR-VL3, X2 is L.

In an alternative embodiment, in CDR-VL3, X2 is V.

In an alternative embodiment, in CDR-VL3, X2 is I.

In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-55:

in alternative embodiments, the antibody or functional fragment thereof binds AMH with K_D≤2.6×10^-8Affinity binding in mol/L.

In an alternative embodiment, K_D≤2×10^-8mol/L、K_D≤1×10^-8mol/L、K_D≤9×10^-9mol/L、K_D≤8×10^-9mol/L、K_D≤7×10^-9mol/L、K_D≤6×10^-9mol/L、K_D≤5×10^-9mol/L、K_D≤4×10^-9mol/L、K_D≤3×10^-9mol/L、K_D≤2×10^-9mol/L or K_D≤1×10^-9mol/L。

In an alternative embodiment, K_D≤9×10^-9mol/L。

In an alternative embodiment, 1.52 × 10^-9mol/L≤K_D≤8.94×10^-9mol/L。

K_DThe detection of (2) is carried out with reference to the method in the examples of the present invention.

In alternative embodiments, in CDR-VH2, X1 is L; in CDR-VH3, X1 is A; in CDR-VL1, X3 is L; in CDR-VL2, X2 is A; in CDR-VL3, X1 is R.

In alternative embodiments, each complementarity determining region of the antibody, or functional fragment thereof, is selected from any one of the following combinations of mutations 56-62:

in alternative embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L in sequence as set forth in SEQ ID NOS: 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H in sequence as set forth in SEQ ID NOS: 5-8.

In general, the variable regions of the heavy chain (VH) and light chain (VL) can be obtained by linking the CDRs and FRs numbered below in a combined arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

It is noted that in other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein can 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 to the corresponding framework region described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8).

In alternative embodiments, the antibody further comprises a constant region.

In alternative embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO. 10.

In alternative embodiments, the functional fragment is selected from any one of VHH, F (ab ') 2, Fab', Fab, Fv and scFv of the antibody.

Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In another aspect, the present invention provides a reagent or kit for detecting AMH, comprising the antibody or functional fragment thereof according to any one of the above.

In an alternative embodiment, the antibody or functional fragment thereof in the above-described reagent or kit is labeled with a detectable label.

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, one skilled in the art can select a suitable marker according to the detection condition or actual requirement, and whatever marker is used belongs to the protection scope of the present invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, Fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine b (tritc), etc. or analogs thereof), Cy-series dyes and derivatives thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or analogs thereof), Alexa-series dyes and derivatives thereof (e.g., including, but not limited to, Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin, pyrenochrome, and derivatives thereof, Allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

In alternative embodiments, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

In alternative embodiments, the radioisotope includes, but is not limited to²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F。

in alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, lotrine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In another aspect, the present invention provides a nucleic acid molecule encoding the above antibody or functional fragment thereof.

In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.

In another aspect, the present invention provides a recombinant cell comprising the vector described above.

In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing the recombinant cell as described above, and separating and purifying the antibody or functional fragment thereof from the culture product.

Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by separating 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, and this is within the scope of the present invention, regardless of the technique used to prepare the antibody or its functional fragment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the result of reducing SDS-PAGE of the anti-AMH antibody of example 1.

Detailed Description

In order to make the objects, 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TM RACE cThe DNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

1 construction of recombinant plasmid

(1) Antibody Gene preparation

mRNA is extracted from a hybridoma cell strain secreting an anti-AMH antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, the Heavy Chain and Light Chain genes are respectively cloned after colonies are grown, and 4 clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 336bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 366bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(3) Construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and a Light Chain gene fragment of 0.74kb and a Heavy Chain gene fragment of 1.46kb are amplified by a PCR amplification method.

The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on days 3, 5 and 7, and the sampling detection is carried out on day 7.

Diluting AMH antigen to 1 μ g/ml with coating solution, 100 μ L per well, and standing overnight at 4 deg.C; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), and drying at 37 deg.C for 1 hr in each well; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 30 min; washing with the washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a developing solution B (50 muL/hole, containing citric acid, EDTA-2 Na, TMB and concentrated HCl) for 10 min; adding stop solution (50 μ L/well, EDTA-2 Na + concentrated H)₂SO₄) (ii) a OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results showed that the OD of the reaction after the cell supernatant was diluted 1000 times was still greater than 1.0, and the OD of the reaction without the cell supernatant was less than 0.1, indicating that the antibody produced after the plasmid transient transformation was active against the AMH antigen.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50 mu L, DNA 100 mu g/tube, Puv I enzyme 10 mu L, sterile water to 500 mu L, 37 ℃ water bath enzyme digestion overnight; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water when ethanol is completely volatilized, and finally measuring the concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cells/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing manner: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. mu.g of an external control antibody was used as a control, and the electrophoretogram showed two bands, 1 of which Mr was 50KD (heavy chain, SEQ ID NO:14) and the other Mr was 28KD (light chain, SEQ ID NO:13), as shown in FIG. 1 below, after the reducing SDS-PAGE.

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Analysis of the antibody (WT) sequence of example 1 showed that the heavy chain variable region is represented by SEQ ID NO:12, wherein the amino acid sequence of each complementarity determining region in the heavy chain variable region is as follows:

CDR-VH1：G-F-T(X1)-F-S-V(X2)-F-G-M-S；

CDR-VH2：T-L(X1)-S-N-G-G-S(X2)-Y-T-Y-Y-P-D(X3)-S-L(X4)-K-G；

CDR-VH3：A(X1)-R-H-P-R-V(X2)-N-G-Y(X3)-D-G-A-M；

the light chain variable region is shown as SEQ ID NO:11, wherein the amino acid sequences of the complementarity determining regions on the light chain variable region are as follows:

CDR1-VL：A-S-E(X1)-S-I(X2)-D-N-Y-D-L(X3)-S-F-M；

CDR-VL2：A-A-S-N-K(X1)-A(X2)-S；

CDR-VL3：Q-Q-S-R(X1)-E-I(X2)-P-W。

based on the anti-AMH antibody (WT) of example 1, mutations were made in the complementarity determining regions at sites involved in antibody activity, wherein X1, X2, X3, and X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

Antibody binding activity assay in table 1:

coating liquid (main component NaHCO)₃) Diluting AMH antigen to 1 mu g/ml for coating a microplate, wherein each well is 100 mu l, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted AMH monoclonal antibody at a concentration of 100 μ l/well at 37 deg.C for 30-60 min; washing with washing solution for 5 times, and drying; goat anti-mouse IgG-HRP was added, 100. mu.l per well, 3730min at the temperature; washing with washing solution (PBS) for 5 times, and drying; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10 min; adding stop solution (50 μ L/well, containing 0.75 g/EDTA.2Na and 10.2ml/L concentrated H2SO 4); OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2 below.

TABLE 2 Activity data of WT antibodies and mutants thereof

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	-	-	-	-

The data in table 2 show that WT, mutations 1 to 4 are more active than mutations 5 and 6, with mutation 1 being the most active.

(2) Affinity detection of antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

Affinity assay

Using AMC sensors, purified antibodies were diluted to 10ug/mL in PBST and AMH antigen was diluted in PBST with a gradient: 45ug/mL, 22.5ug/mL, 11.3ug/mL, 5.60ug/mL, 2.80ug/mL, 1.40ug/mL, 0.70 ug/mL.

The operation flow is as follows: equilibrating in buffer 1(PBST) for 60s, immobilizing antibody in antibody solution for 300s, incubating in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociating in buffer 2 for 1200s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data. K_DIndicates the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate. The results are shown in Table 4 below.

Table 4 affinity assay data

As can be seen from the data in Table 4, the mutant 1 and the mutants in the series have better affinities, which indicates that the antibodies obtained by mutation in the way of the mutation in Table 3 have better affinities on the basis of the mutant 1.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 affinity assay results for WT antibodies and their mutants

	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

The data in Table 6 show that WT and its series of mutants all have good affinity, indicating that based on WT, the antibodies mutated in the way of the mutations in Table 5 all have good affinity.

(3) Evaluation of stability against naked antibody

Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the antibody is stable. The following table 7 shows the results of the OD detection of the 21-day assessment of the antibody to mutation 1.

TABLE 7

Sample concentration (ng/ml)	100	10	0
				Samples at 4 ℃ for 21 days	1.985	0.528	0.05
21 days samples at-80 deg.C	1.93	0.538	0.045
				21 day samples at 37 deg.C	1.931	0.593	0.051

(4) Application performance detection

The antibody in Table 3 is matched with another strain of AMH antibody for use, and a double-antibody sandwich method is adopted, so that the detection specificity of a chemiluminescence platform reaches 100%, and the sensitivity reaches 99% -99.8%.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Dongguan City Peng Zhi Biotech Co., Ltd

<120> antibody against AMH, reagent and kit for detecting AMH

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<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> Artificial 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> Artificial 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 or functional fragment thereof directed against AMH, wherein said antibody or functional fragment thereof comprises the following complementarity determining regions:

CDR-VH 1: G-F-X1-F-S-X2-F-G-M-S; wherein: x1 is S or T; x2 is I, V or L;

CDR-VL 2: A-A-S-N-X1-X2-S; wherein: x1 is K, Q or R; x2 is G or A;

CDR-VL 3: Q-Q-S-X1-E-X2-P-W; wherein: x1 is K and R; x2 is L, V or I.

2. The anti-AMH antibody, or functional fragment thereof, according to claim 1,

in CDR-VH2, X1 is I;

in CDR-VH3, X1 is T;

in CDR-VL1, X3 is I;

in CDR-VL2, X2 is G;

in CDR-VL3, X1 is K;

preferably, in CDR-VH1, X1 is S;

preferably, in CDR-VH1, X1 is T;

preferably, in CDR-VH1, X2 is I;

preferably, in CDR-VH1, X2 is V;

preferably, in CDR-VH1, X2 is L;

preferably, in CDR-VH2, X2 is T;

preferably, in CDR-VH2, X2 is S;

preferably, in CDR-VH2, X3 is E;

preferably, in CDR-VH2, X3 is D;

preferably, in CDR-VH2, X4 is I;

preferably, in CDR-VH2, X4 is V;

preferably, in CDR-VH2, X4 is L;

preferably, in CDR-VH3, X2 is I;

preferably, in CDR-VH3, X2 is V;

preferably, in CDR-VH3, X2 is L;

preferably, in CDR-VH3, X3 is Y;

preferably, in CDR-VH3, X3 is F;

preferably, in CDR-VH3, X3 is S;

preferably, in CDR-VL1, X1 is Q;

preferably, in CDR-VL1, X1 is E;

preferably, in CDR-VL1, X2 is I;

preferably, in CDR-VL1, X2 is V;

preferably, in CDR-VL1, X2 is L;

preferably, in CDR-VL2, X1 is K;

preferably, in CDR-VL2, X1 is Q;

preferably, in CDR-VL2, X1 is R;

preferably, in CDR-VL3, X2 is L;

preferably, in CDR-VL3, X2 is V;

preferably, in CDR-VL3, X2 is I;

preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-55:

3. the antibody or functional fragment thereof against AMH according to claim 2, wherein said antibody or functional fragment thereof binds to the anti-AMH antigen with K_D≤2.6×10^-8Affinity binding of mol/L; preferably, K_D≤9×10^-9mol/L。

4. The anti-AMH antibody or functional fragment thereof according to claim 1,

in CDR-VH2, X1 is L;

in CDR-VH3, X1 is A;

in CDR-VL1, X3 is L;

in CDR-VL2, X2 is A;

in CDR-VL3, X1 is R;

preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 56-62:

5. the antibody or functional fragment thereof against AMH according to any one of claims 1 to 4, wherein said antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, having the sequence shown in SEQ ID NO 1-4 in order, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, having the sequence shown in SEQ ID NO 5-8 in order;

preferably, the antibody further comprises a constant region;

preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the constant region is from a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fight, or human;

preferably, the constant region is derived from a mouse;

preferably, the light chain constant region sequence of the constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the constant region is shown as SEQ ID NO. 10;

preferably, the functional fragment is selected from any one of VHH, F (ab ') 2, Fab', Fab, Fv and scFv of the antibody.

6. A reagent or kit for the detection of AMH, comprising an antibody or functional fragment thereof according to any one of claims 1 to 5.

7. The reagent or kit according to claim 6, wherein the antibody or functional fragment thereof is labeled with a detectable label;

preferably, the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label;

preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;

preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;

preferably, the radioisotope is selected from the group consisting of²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F；

preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lowhine and its derivatives, and peroxyoxalate and its derivatives;

preferably, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes;

preferably, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver and colloidal selenium.

8. A vector comprising a nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1 to 5.

9. A recombinant cell comprising the vector of claim 8.

10. A method for producing an antibody or functional fragment thereof according to any one of claims 1 to 5, comprising: culturing the recombinant cell of claim 9, and isolating and purifying the antibody or functional fragment thereof from the culture product.