The invention relates to the field of in vitro detection of anti-Müllerian hormone called AMH otherwise. In particular, the invention relates to the preparation of anti-AMH antibody, anti-AMH antibodies and their use in determining the concentration of AMH, particularly in the context of investigations related to fertility in women or female animals of childbearing age.
The anti-Mullerian hormone (AMH also called for anti-Mullerian hormone) is a dimeric glycoprotein of 144 kDa of the family of transforming growth factor (TGF-β) family with many factors affecting the growth and differentiation. AMH is a dimeric prohormone: it consists of two identical subunits which are linked by disulfide bridges. This prohormone undergoes proteolytic cleavage near the C-terminus to acquire biological activity and develop into mature hormone. After cleavage, the molecular complex remains associated and is measurable in the blood by immunoassay suitable for sensitivity performance in monitoring the evolution of ovarian reserve from birth to menopause (KELSEY TW, et al, 2011). This protein is also known as MIF (Mullerian-inhibiting factor), MIH (Mullerian-inhibiting hormone) and MIS (Mullerian-inhibiting substance).

AMH is present in all mammals. Its length and amino acid sequence are dependent on the species. Thus, human ΓΑΜΗ has 560 amino acids and is composed as follows: a short signal peptide (amino acids 1-18), a precursor part (amino acids 19-25), an N-terminal portion (amino acids 26-451) and a C-terminal part (amino acids 452-560). Equine AMH has 573 amino acids and is composed as follows: a short signal peptide (amino acids 1-22), no precursor portion, a N-terminal part (amino acids 23-464) and a C-terminal portion ( amino acids 465-573). Canine AMH has 572 amino acids and is composed as follows: a short signal peptide (amino acids 1-21), no precursor portion, a N-terminal part (amino acids 22-463) and a C-terminal portion ( amino acids 464-572). Bovine AMH in turn has 575 amino acids and is composed as follows: a short signal peptide (amino acids 1-17), a precursor part (amino acids 18-24), an N-terminal part (amino acids 25- 466) and a C-terminal part (amino acids 467-575). Whatever the case, the N-terminal part of ΓΑΜΗ is called "pro region" and the C-terminal part is called "mature region."

AMH is synthesized as a precursor polypeptide comprising the signal peptide, followed by the pre-prohormone (humans and cattle) or prohormone (in the horse or dog). The signal peptide, which allows targeting to the endoplasmic reticulum, is cleaved after translocation. In the endoplasmic reticulum, the precursor polypeptide undergoes post-translational modifications, namely (i) the homodimerization and (ii) glycosylation, in order to achieve its native conformation. In humans, it is a dimeric glycoprotein of 144 kDa corresponding to amino acids 26-560. Each monomer of 72 kDa in humans and contains the pro region (58 kDa in humans) followed by the mature region (12 kDa in humans). Before secretion into the extracellular medium and the blood circulation, AMH glycoprotein ultimate undergoes post-translational processing cleavage. Thus, humans and Cattle, the pre-prohormone is converted into prohormone cleavage of the precursor portion. The pro-hormone is also cleaved from the N-terminal part and the C-terminal portion to obtain the mature hormone (having a biological activity). These two parts but remained non-covalently linked (Zec I., et al, 2011). In humans, varying cleavage rates have been reported in different studies: between 5 to 20% by Zec et al. and 67% in boys or 81% in women by Pankhurst et al. (2016). Thus, in the bloodstream include ΓΑΜΗ of cleaved and uncleaved ΓΑΜΗ.

AMH is produced in the male by the Sertoli cells and occurs at the beginning of his life to the differentiation of the male tract. The female ΓΑΜΗ is produced by the granulosa cells of small growing follicles and thus comes rather from puberty. Thus, in women, ΓΑΜΗ has shown its utility in various fields, particularly related to fertility (DEWAILLY D, et al. 2014). For example, the metering ΓΑΜΗ in circulation makes it possible to estimate the number of antral follicles and pre-antral present in the ovaries, regardless of the cycles (DEWAILLY D, et al, 2014). In addition, as an indicator of the natural decay of ovarian reserve and thus the risks of infertility, the dosing

ΓΑΜΗ shown to help women manage their pregnancy plans. In a context of assisted reproduction, dosage AMH helps to select the best strategy for the patient, in particular by optimizing the controlled ovarian stimulation stage while avoiding the risk of hyperstimulation (ARCE JC, et al, 2014 ). The dosage AMH also tracks the evolution of ovarian reserve in young girls or women who received gonadotoxic treatment for cancer, for example (CHAI J and HOWIE AF., 2014). For women with ovulation disorders, determination of serum AMH to better characterize the type of ovarian dysfunction, particularly hypergonadotropique anovulation associated with ovarian failure as polycystic ovary syndrome (FONG SL and al, 2015). In young boy ΓΑΜΗ occurred dramatically by the testicles of the fetus and newborn and thus intervenes at an early stage in the differentiation of the male tract. Thus, the dosage ΓΑΜΗ found utility in boys before puberty, in the context of disorders of sexual differentiation.

The AMH assay is performed by sandwich immunoassay and requires the implementation of a test both sensitive, specific and reproducible. However, despite the various methods proposed in the literature (HUDSON, 1990. LONG, 2000) or the various kits available on the market, an assay that combines all of these features is still not available. A major difficulty in solving was identified in 1996: this is the apparent lack of stability of SEP in the biological sample. Thus, the storage conditions (time, temperature, freezing cycles, ...) can cause variations in the measured concentrations (Lee, 1996). These variations are of course an artifact and may result in misinterpretation of the biological result.

Different kits have been placed on the market. The antibodies used in these kits recognize different regions of ΓΑΜΗ. The first generation of AMH assays corresponds to EIA kits MIS / AMH (Immunotech) and Active kit MIS / AMH ELISA (DSL). The antibodies used in these kits are not identical but each uses a first antibody recognizing the pro region of ΓΑΜΗ, another antibody recognizing the mature region. In 2010, Beckman Coulter company which marketed them, replaced them with a second generation kit (AMH Gen II Assay) with, according to them, improved performance. According to the authors, this kit is highly specific and measured ΓΑΜΗ would be unaffected by proteolysis because the two monoclonal antibodies used each recognize the mature region (Kumar, 2015; US7897350). The authors believe that the mature region, which contains several cysteine ​​residues would be more stable than the pro region if proteolysis. When deployed on the ground, in routine clinical use, it soon became clear that the AMH Gen II kit did not keep its promises. A large retrospective study confirmed the lack of stability ΓΑΜΗ dosage given the conditions of storage of sample tubes (O Rustamov, 2012) and showed that the kit AMH Gen II was extremely sensitive to these variations. Several hypotheses have been advanced to explain the observed fluctuations. One interpretation could be related to the fact that the molecule AMH undergoes conformational changes after removal of a blood sample, variable from one individual to another.

These results were quickly repeated and confirmed by an independent team (XAN et al, 2014). In addition, the team describes a way to promote stability ΓΑΜΗ during sample storage: they carried out a pre-dilution prior to dosing, thereby improving the overall reproducibility of assays AMH Gen II . This solution certainly easy to implement, however, a few drawbacks:

(I) The AMH concentrations measured after pre-dilution are increased. This increase varies from one individual to another (from 1.35 times to 3.06 times for the available data). It is therefore necessary to redefine the reference values ​​experimentally on a large cohort and clinical interpretation of the rules.

(Ii) The dilution carried out is not negligible (1/5, 60 of sample into 300 of buffer). It certainly eliminates the observed interference, but actually causes a decrease in the sensitivity of the assay, important for many clinical uses.

(Iii) because of the instability of ΓΑΜΗ still not identified. By

Therefore, it is not possible to assert that dilution could solve the problem of lack of stability for all samples.

Moreover, Lukaszuk et al. (2014) described a reduction in the average concentration of AMH measured by the AMH Gen II Assay kit and a decrease in sensitivity. Their conclusion is that it is "very dangerous to implement a stimulation protocol based on the results of the Gen II kit."

It is therefore essential to have a robust package, for which the concentration of AMH measured is correct and reproducible, regardless of the time between sampling and dosing, and regardless of the storage conditions (independent dose conditions preanalytical).

Patent application WO2014 / 074835 proposes different assay methods of different isoforms AMH (AMH dimeric only, not cleaved AMH, AMH cleaved then re-associated, ...) to determine whether a particular form iso or changing a isoform to another can have a clinical sense. Thus, the application describes many antibodies directed against a large number of linear epitopes, located either in the pro region or in the mature region of ΓΑΜΗ. The authors claim that the compositions and methods disclosed fulfill the need for a right balance, reproducible and standardized. This application discloses 18 epitopes, in total 324 (18x18) ways to build sandwich immunoassay combining these antibodies recognizing epitopes. Given the many pre-analytical problems encountered with different dosages of ΓΑΜΗ, it is difficult to believe without experimental evidence that each of these epitope combinations therefore antibody, allows to obtain a robust test. Moreover, no data in this patent application shows that the use of the antibodies described allows a usable dosing in primary care, robust, accurate, reproducible and standardized, and above all, the result is independent of the pre-analytical conditions. The only data presented in the application relate to stability data from type assays "single sandwich or ITS epitope", namely assays using the same monoclonal antibody capture and detection. In addition, these tests detect only the dimeric form of ΓΑΜΗ whose clinical significance is yet unknown (as measured concentrations are very

various concentrations obtained by the total AMH assays which are those used in current medical practice).

The Applicant has unexpectedly found that it was possible to overcome the disadvantages of the prior art by preparing new antibodies recognizing that non-linear epitopes in a particular area of ​​ΓΑΜΗ located in the middle part of the pro region of SEP (between amino acids 157-255 in humans), which antibodies can perform a stable and reliable AMH dosage used in medical practice, robust, fair and reproducible, regardless of pre-analytical conditions or sample storage. By choosing this particular region of AMH, described as having a cleavage region (WO2014 / 074835), the metering ΓΑΜΗ with these nonlinear epitope recognition antibody on the defined area detects Surprisingly and against all expectations any Γ AMH of biological interest present in a biological sample.

Thus, a first object of the invention relates to a method of preparing mammalian anti-antibody AMH (Anti-Mullerian hormone mammalian), characterized in that it comprises the steps of:

(I) immunizing an animal with a polypeptide of AMH or a polynucleotide encoding the polypeptide of AMH, AMH said polypeptide comprising at least 99 amino acids of sequence SEQ ID NO: l or sequence having at least 75% identity with the sequence SEQ ID No. 1, and at most 560 amino acids of sequence SEQ ID NO: 2 or sequence having at least 75% identity with the sequence SEQ ID No. 2,

(Ii) preparing hybridomas from cells of a lymphoid organ of the animal that received the immunogen,

(Iii) selecting the hybridomas secreting antibodies recognizing a AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID No. 1 or sequence having at least 75% identity with the sequence SEQ ID NO: l, and at most 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID N ° 8, but not recognizing either (a) a polypeptide comprising at least the AMH 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID No. 11 or of sequence having at least 75% identity to SEQ ID no 11, or (b) no linear epitope in SEQ ID no 1 or a sequence having at least 75% identity with the sequence SEQ ID nO: l, and

(Iv) production of antibodies.

A second object of the invention relates to monoclonal or fragments of mammalian anti-AMH antibody monoclonal antibody recognizing a polypeptide of AMH comprising at least 99 amino acids of sequence SEQ ID No. 1 or sequence having at least 75% identity with the sequence SEQ ID nO: l and at most 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID nO: 8, but recognizes neither (a) a polypeptide AMH comprising at least the 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence SEQ ID N ° 13 and at most 156 amino acid sequence SEQ ID No. 11 or of sequence having at least 75% identity with the sequence SEQ ID No. 11 or any linear epitope located within the sequence SEQ ID No. 1 or a sequence having at least 75% identity with the sequence SEQ ID No. l.

A third object of the invention relates to conjugates comprising (i) a monoclonal antibody or a fragment of anti-AMH monoclonal antibody as defined above or prepared by the process as defined above and (ii) a marker capable of generating the emission of a detectable signal for displaying an immunological reaction between said conjugate and ΓΑΜΗ a biological sample.

A fourth object of the invention relates to a AMH quantization process mammal by sandwich immunoassay, in a biological sample likely to contain ΓΑΜΗ, which comprises the steps of:

- contacting said biological sample with two binding partners ΓΑΜΗ at least one of said partner is an antibody or antibody fragment such as defined above or as prepared according to the method as defined above, or a conjugate as defined above,

- detecting a signal transmitted by the binding between said binding partners ΓΑΜΗ and, if present, using a label capable of emitting a detectable signal, and - converting the detected signal into a concentration of AMH.

A fifth object of the invention concerns the use of a method of quantifying AMH as defined above as an aid for the diagnosis of disorders associated with ovarian dysfunction in women of childbearing age or as an aid evaluation of ovarian follicular reserve in girls over 12 years and women, or as an aid in the evaluation of disorders of sexual differentiation in boys before puberty.

Finally, a last object of the invention relates to a kit comprising an antibody or antibody fragment such as defined above or as prepared according to the method as defined above, or a conjugate as defined above .

The Applicant has therefore unexpectedly found that the use of the median part of the pro region of SEP for selecting anti-AMH monoclonal antibodies during their preparation process, while avoiding the linear epitopes recognized antibody of this part, allowed when those thus selected antibodies are used for the determination of AMH, to have a reliable and reproducible dose.

An epitope, also known as antigenic determinant, is the smallest part of an antigen that can be recognized by a paratope which is the variable portion of an antibody. The structure of the epitope is complementary with the paratope of the antibody. The structure set game may be the primary structure, in the case of a linear epitope, sequential epitope or also known as continuous epitope comprising consecutive amino acids or the tertiary structure in the case of a discontinuous epitope, also known as epitope non-linear. Where the antigen is protein in nature, as in this case, linear epitopes correspond to a peptide sequence of variable length.

The sequence of a linear epitope may include so-called conservative changes which do not change significantly the bond between the epitope and the antibody from a viewpoint of specificity.

The antibodies prepared according to the method of the invention therefore do not recognize linear epitopes on the middle part of the pro region SEP but recognize this middle part.

By monoclonal antibodies recognizing the median part of the pro region of ΓΑΜΗ is meant that the antibodies are capable of binding to ΓΑΜΗ when this portion is present, such as full ΓΑΜΗ, but are not capable of binding to a polypeptide AMH when this part is absent.

Without being bound by theory, a hypothesis a posteriori would suggest that the middle part of the pro region of ΓΑΜΗ against which antibodies are directed may correspond to an area where ΓΑΜΗ would not suffer proteolysis in the biological sample in which it is measured, for example plasma, so that this area remain well presented in its original conformation, or that its shape would not be affected by changes that could affect the surrounding areas of the molecule.

Mammals to which it is desired to produce antibodies against their middle part of the pro region of AMH their are any mammal for which the determination of AMH constitute aid. Examples include human (woman and man the boy), the Horse (especially the mare), the dog (especially the dog), the cattle, the sheep, the Felines.

The AMH secreted by mammals are not identical but are close. Between human AMH ΓΑΜΗ and other mammals for which the dosage AMH constitutes aid, at least 75% sequence identity is observed. AMH taken Reference herein will ΓΑΜΗ human.

The "identity" or "percent identity" in the context of two or more polypeptide sequences mean that the two compared sequences have a specified percentage of amino acid residues that are the same on the maximum length that could be aligned , when aligned for maximum correspondence. Such alignments and calculation of percent identity can be achieved by algorithms and / or sequence comparison programs (Emboss Needle, LALIGN, Blast, Clustal, ...) or by visual inspection in simple cases.

As indicated above, ΓΑΜΗ comprises a pro region and a mature region. In humans, the pro region ends at position 451 and the mature region begins at position 452. The middle part of the pro region of ΓΑΜΗ recognized by antibodies prepared according to the invention begins in humans

position 157 and ends at position 255. It consists of 99 amino acids of the sequence SEQ ID No. l. In the horse, the middle portion of the ΓΑΜΗ pro region recognized by the antibodies prepared according to the invention starts at position 167, ends at position 265 and corresponds to 99 acid sequence SEQ ID NO: 14. The middle part of the pro region of canine SEP recognized by antibodies prepared according to the invention begins in turn at position 166, ends at position 264 and corresponds to the sequence of 99 acids SEQ ID NO: 23. With regard to the cattle, the middle portion of the ΓΑΜΗ pro region recognized by the antibodies prepared according to the invention starts at position 171, ends at position 270 and 100 corresponds to the sequence of acids SEQ ID NO: 32.

To produce these anti-AMH antibody individual and advantageous, the method of the invention comprises as a first step, immunization of an animal with an immunogen. This immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which must include at least the region that the antibody should recognize, namely at least the middle portion of the pro region of ΓΑΜΗ. Thus, the immunogen used comprises at least the 99 amino acids of SEQ ID NO sequence or the sequence having at least 75% identity with the sequence SEQ ID No. 1.

The immunogen also comprises at most ΓΑΜΗ whole of the species in question or the entire polynucleotide encoding ΓΑΜΗ of the species, i.e., in the case of human ΓΑΜΗ, the 560 amino acids of sequence SEQ ID No. 2 (N Uniprot accession ° F2YMM5 KB), other AMH comprising at least 75% identity with SEQ ID NO: 2 sequence. For example, the polypeptide total equine AMH 573 comprises the amino acid sequence SEQ ID No. 15 (Accession No. Uniprot F2YMM5 KB), the polypeptide of total canine AMH 572 comprises the amino acid sequence SEQ ID No. 24 (Uniprot accession No. A0A0E3N0I3 KB) and the total bovine AMH polypeptide (Uniprot accession No. P03972 KB) comprises the 575 amino acids of sequence SEQ ID N ° 33.

According to a particular embodiment, the immunogen used is a polypeptide of AMH, or polynucleotide encoding such polypeptide devoid, in relation to the entire AMH sequence, all the amino acids located after the middle part of the region pro ΓΑΜΗ, namely a polypeptide of AMH, or a polynucleotide encoding said polypeptide AMH, comprising at least the 230 amino acid sequence SEQ ID No. 10 or of sequence having at least 75% identity with the sequence SEQ ID N ° 10 and at most 255 amino acids of sequence SEQ ID NO 8 or sequence having at least 75% identity with the sequence SEQ ID No. 8, preferably selected from polypeptides of AMH SEQ ID NO: 8, SEQ ID No. 9 and SEQ ID NO: 10 or sequences having at least 75% identity with these sequences. SEQ ID NO: 8 is amino acids 1-255 of human ΓΑΜΗ (called AMH-2), the sequence SEQ ID No. 9 corresponds to amino acids 19-255 of human ΓΑΜΗ, namely SEQ ID NO sequence 8 without the signal (called AMH-2 without signal peptide) peptide and the sequence SEQ ID nO: 10 corresponds to amino acids 26-255 of human ΓΑΜΗ, namely the sequence SEQ ID N ° 8, without the signal peptide, or the part precursor (called AMH-2 peptide precursor without signal or part).

According to another embodiment, the immunogen used is a polypeptide of AMH, or polynucleotide encoding such polypeptide, which corresponds to total ΓΑΜΗ, with all or part of the signal peptide and optionally all or part of the precursor part. Thus, the polypeptide of AMH comprises at least the 535 amino acid sequence SEQ ID NO: 4 or sequence having at least 75% identity with the sequence SEQ ID NO: 4, preferably selected from polypeptides AMH sequences SEQ ID No. 2, SEQ ID NO: 3 and SEQ ID No. 4 or sequences having at least 75% identity with these sequences. SEQ ID NO: 2 corresponds to amino acids 1-560 of human SEP, the sequence SEQ ID NO 3 corresponds to amino acids 19-560 of human ΑΜΗ, namely SEQ ID No. 2 without the signal peptide, and the sequence SEQ ID nO 3 corresponds to amino acids 26-560 of human ΑΜΗ, namely SEQ ID No. 2 without the signal peptide, or the precursor part.

According to another embodiment, the immunogen used is a polypeptide of AMH, or polynucleotide encoding such polypeptide, which corresponds to total ΑΜΗ, with all or part of the signal peptide and optionally all or part of the precursor portion, but without the mature region. Thus, the polypeptide of AMH comprises at least the 426 amino acid sequence SEQ ID NO: 7 or sequence having at least 75% identity with the sequence SEQ ID N ° 7 and at most 451 amino acids of sequence SEQ ID No. 5 or sequence having at least 75% identity with the sequence SEQ ID NO: 5, preferably selected from polypeptides AMH sequences SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO 7 or sequences having at least 75% identity with these sequences. SEQ ID NO: 5 sequence corresponds to amino acids 1-451 of human ΓΑΜΗ (called AMH-3), the sequence SEQ ID No. 6 corresponds to amino acids 19-451 of human ΓΑΜΗ, namely SEQ ID NO sequence 5 without the signal (called AMH-3 without signal peptide) peptide and the SEQ ID N ° 7 sequence corresponds to amino acids 26-451 of human ΓΑΜΗ, namely SEQ ID nO: 5 without the signal peptide, or the part precursor (called AMH-3 peptide precursor without signal or part).

The following table (Table 1) has different AMH useful polypeptides in the context of the invention, in particular as an immunogen for some, giving the positioning of the amino acids and the corresponding SEQ ID (in parentheses) according to mammalian species.

Table 1

Humaine Equine Canine Bovine

Midsection

157-255 167-265 166-264 171-270 in the region

(SEQ ID N°l) (SEQ ID N°14) (SEQ ID N°23) (SEQ ID N°32) Pro de l'AMH

1-560 1-573 1-572 1-575

raw totale

(SEQ ID N°2) (SEQ ID N°15) (SEQ ID N°24) (SEQ ID N°33)

raw totale

19-560 23-573 22-572 18-575 without peptide

(SEQ ID N°3) (SEQ ID N°16) (SEQ ID N°25) (SEQ ID N°34) signal

raw totale

without 26-560 25-575 peptide

NA NA

signal, or part (SEQ ID No. 4) (SEQ ID NO: 35) precursor

1-451 1-464 1-463 1-466

No. 3-totale

(SEQ ID N°5) (SEQ ID N°17) (SEQ ID N°26) (SEQ ID N°36)

No. 3-totale

19-451 23-464 22-463 18-466 without peptide

(SEQ ID N°6) (SEQ ID N°18) (SEQ ID N°27) (SEQ ID N°37) signal

No. 3-totale

without 26-451 25-466 peptide

NA NA

signal, or portion (SEQ ID NO: 7) (SEQ ID NO: 38) precursor

1-255 1-265 1-264 1-270

No. 2-totale

(SEQ ID N°8) (SEQ ID N°19) (SEQ ID N°28) (SEQ ID N°39)

No. 2-totale

19-255 23-265 22-264 18-270 without peptide

(SEQ ID N°9) (SEQ ID N°20 (SEQ ID N°29) (SEQ ID N°40) signal

No. 2-totale

without 26-255 25-270 peptide

NA NA

signal, or portion (SEQ ID NO: 10) (SEQ ID No. 41) precursor

NA = Not Applicable because no part precursor in ΓΑΜΗ concerned

Of course, besides the amino acids described in these sequences, the immunogen, when it is in the form of polypeptide, can also comprise other amino acids used for production of the polypeptide or its purification, such as a polyhistidine tail.

The polypeptide production methods are widely known in the art. For example, polypeptides AMH can be obtained by genetic engineering using the steps, conventionally known to those skilled in the art, of:

- have the DNA encoding the polypeptides of AMH, said DNA being obtained according to conventional methods,

- insert this DNA by cloning into an expression vector such as a plasmid, cosmid, phage λ, or a viral vector (baculovirus (Autographa californica Nuclear Polyhedrosis Virus) virus, vaccinia, Semliki Forest virus, adeno virus, lentivirus, ...), which vector also includes an origin of replication (for plasmids or cosmids) or replication system allowing its amplification in the host cell and one or more promoters for the transcription of messenger RNA to be translated protein,

- introducing the vector with the gene of interest for expression in a host cell, such as a prokaryotic cell (e.g., bacteria such as Escherichia coli, Bacillus subtilis) by transformation or infection or a eukaryotic cell (e.g. yeast cells (Saccharomyces cerevisiae, Pichia pastoris), insect cells (Sf9, Sf21, High5), mammalian cells (CHO, 293, Per.C6, BHK-21, Vero ...) by transient or permanent transfection, or viral infection,

- culture and optionally propagating the host cell containing the expression vector, optionally with amplification of the vector in the host cell,

- where necessary, inducing transcription and protein synthesis for the production of recombinant polypeptides AMH, and

- purifying said polypeptides to extract, for example by a polyhistidine tail. The polypeptides are said to recombinants.

When the immunogen is a polynucleotide, for example, use the same DNA as that which would be used to prepare the polypeptide by genetic engineering AMH.

The animal used for immunization is usually any animal used to obtain monoclonal antibodies, such as for example a mouse, rat, rabbit, goat or sheep.

The conditions and parameters for immunization, such as immunogenic concentration, mode of immunization, etc. are widely known to the skilled person that can refer to the procedures described by Kohler and Milstein in 1975, particularly in Current protocols in Cell Biology Manual (WM Yokoyama, 2001).

The second step of the process of the invention is a conventional step and is to prepare hybridomas from cells of a lymphoid organ of the animal that received the immunogen, such as for example the spleen, lymph nodes and

tonsils. Such a step is described in any manual of generating monoclonal antibodies. However, use the fusion partner for every species. Thus, for example, mice, can be used to line cGPS CHO-Sa for the rabbit of the 240E-W line (US7,429,487) and the sheep's SFP1 line (W092 / 15699).

The third step of the method consists in the particular selection of resultant hybridomas. To do this, the hybridomas are first cultured in a suitable medium, such as well-known to the skilled person, including in particular nutrients for their propagation, growth stimulators. Then selecting clones secreting the antibody of interest is performed using the antigen recognized by the antibody according to human techniques known in the art such as the direct detection without markers (spontaneous precipitation after antigen-antibody reaction), the indirect detection (e.g., ELISA or other type of immunoassay), the surface plasmon resonance (BIAcore®) and interferometry (Byte). The novelty of this step is the particular selection of hybridomas that can be implemented in two successive screens, in any order, with individual polypeptides and peptides.

The first screening, called screening 1, is to retain only the hybridomas that produce antibodies that recognize the middle part of the pro region, as described above, area is known for yet have a cleavage site and instead being excluded. To do this, two methods are possible:

Method 1:

- firstly are selected hybridomas secreting antibodies recognizing SEP-2, i.e., to human SEP-2, 1-255 amino acids (SEQ ID NO: 8), where appropriate without all or part of the signal peptide and all or part of the precursor portion, or any sequence having at least 75% identity with this sequence. According to one embodiment, the AMH-2 polypeptide recognized by the antibody is a sequence polypeptide selected from: SEQ ID No. 8, SEQ ID No. 9 and SEQ ID NO: 10 and sequences having at least 75% identity with these sequences.

- Then one removes the hybridomas which produce antibodies recognizing the part of ΓΑΜΗ located before the middle part of the pro region of ΓΑΜΗ, namely,

for human ΓΑΜΗ, amino acids 1-156 (SEQ ID NO: 11), where appropriate without all or part of the signal peptide and part or all of the precursor portion, and any sequence having at least 75% identity with this sequence. This polypeptide is referred AMH AMH polypeptide (a) not recognized by the antibodies prepared according to the invention. According to one embodiment, the polypeptide of AMH (a) not recognized by the antibody is a polypeptide sequence selected from: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and sequences having at least 75% identity with these sequences. SEQ ID NO: 11 corresponds to the 1-156 amino acids of human ΓΑΜΗ (called AMH-1), SEQ ID N ° 12 sequence corresponds to amino acids 19-156 of human ΓΑΜΗ, namely SEQ ID NO: 11 without the signal peptide (referred AMH-1 without signal peptide) and the sequence SEQ ID nO: 13 corresponds to amino acids 26-156 of human ΓΑΜΗ, namely the sequence SEQ ID N ° 11 without the signal peptide, or the part precursor (called AMH-1 without signal peptide precursor or part).

Method 2:

- is selected hybridomas secreting antibodies that recognize the middle part of the pro region of ΓΑΜΗ, namely for human ΓΑΜΗ, amino acids 157-255 (SEQ ID NO: 1) and any sequence having at least 75% identity with this sequence.

The table below (Table 2) has different polypeptides AMH (a) are not recognized, useful in the context of the invention, giving the positioning of the amino acids and the corresponding SEQ ID (in parentheses) depending on the species mammalian.

Table 2

NA = Not Applicable because no part precursor in ΓΑΜΗ concerned

Thus, in a particular embodiment, the sequences having at least 75% identity with SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 13 are selected from the sequences: SEQ ID N 21, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

Polypeptides AMH (a) may be prepared as described above and comprise other amino acids not belonging to ΓΑΜΗ.

The second screening, called screening 2 is to separate the hybridomas which produce antibodies which recognize linear epitopes in the middle part of the pro region of ΓΑΜΗ, namely in SEQ ID NO: sequence I or any sequence having 75%> d identity with the sequence SEQ ID No. 1. These epitopes are called linear epitopes (b).

The determination of linear epitopes on a sequence of amino acids is well known to those skilled in the art. It consists in cutting out the sequence of interest into overlapping peptides of predetermined length, for example 10, 12 or 15 amino acids, then identify among these peptides those recognized by monoclonal or polyclonal antibodies. Of course, by their definition, linear epitopes have consecutive amino acid sequence of interest.

The table below (Table 3) present different linear epitopes (b)

determined in the middle part of the pro region of ΓΑΜΗ useful in the context of the invention, giving SEQ ID according to the mammalian species.

Table 3

Thus, in one embodiment, linear epitopes (b) not recognized by the antibodies have the sequences SEQ ID NO: 45 to SEQ ID NO: 56.

As previously, the epitopes are produced by conventional methods, as described above. They can also be produced by peptide synthesis, which is often preferred.

A particular embodiment comprises at least the following characteristics to the selection step (iii):

Screening 1 implements the method 1;

Screening 1 is carried out prior to screening 2.

Between each round of screening, if necessary, the antibodies contained in the hybridoma supernatants were purified according to conventional techniques, such as affinity chromatography on protein A. After the selection and / or desired, between each round of screening, hybridomas are cloned to ensure a steady and homogeneous line. This is widely known to those skilled in the art.

The last process step of the invention involves the production of antibodies of interest, also well known step in the art and described in all of the production of monoclonal antibodies manuals, such as Current Protocols in manual Cell Biology.

In addition to the above steps, including the general procedure has been widely described, the method of the invention may also comprise other steps such as washing, preservation and processing of the antibody fragments.

In the method for preparing anti-AMH antibody of the invention, the immunogen (AMH polypeptide or the polynucleotide encoding the polypeptide comprising at least 99 amino acids of sequence SEQ ID No. 1 or having at least sequence 75% identity with the sequence SEQ ID NO: l and at most 560 amino acids of sequence SEQ ID NO: 2 or sequence having at least 75% identity with the sequence SEQ ID No. 2), the middle part of the pro region of ΓΑΜΗ (AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID No. 1, or sequence having at least 75% identity with the sequence SEQ ID NO: l, and plus 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID No. 8), the polypeptide (a) not recognized by the antibody of interest (polypeptide of AMH comprising at least the 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence S EQ ID NO: 13 and at most 156 amino acids of sequence SEQ ID NO: ll or sequence having at least 75% identity with the sequence SEQ ID No. 11) and the linear epitope (located within the sequence SEQ ID No. 1 or a sequence having at least 75% identity with the sequence SEQ ID NO: l) preferably belong to the same species.

According to one embodiment:

- the mammal is human, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 99 amino acids of sequence SEQ ID N ° 1 and at most 560 acids amino sequence SEQ ID NO: 2, the polypeptide of AMH recognized by the antibodies secreted by hybridomas is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID NO: l and at most 255 amino acids sequence SEQ ID No. 8, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 131 amino acids of sequence SEQ ID N ° 13 and at most 156 amino acid sequence SEQ ID NO: 1, or

- the mammal is a horse, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least 99

amino acid sequence SEQ ID N ° 14 and at most 573 amino acids of sequence SEQ ID N ° 15, the AMH polypeptide recognized by antibody secreted by the hybridoma is an AMH polypeptide comprising at least the 99 amino acids sequence SEQ ID N ° 14 and at most 265 amino acids of sequence SEQ ID N ° 19, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least the 144 amino acids of sequence SEQ ID N ° 22 and at most 166 amino acids of sequence SEQ ID No. 21, or

- the mammal is a dog, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 99 amino acids of sequence SEQ ID N ° 23 and at most 572 amino acids sequence SEQ ID N ° 24, the AMH polypeptide recognized by antibody secreted by the hybridoma is an AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID N ° 23 and at most 264 amino acid sequence SEQ ID nO: 28, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 144 amino acids of sequence SEQ ID N ° 31 and at most 165 acids amino sequence SEQ ID NO: 30, or

- the mammal is a bovine, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 100 amino acids of sequence SEQ ID No. 32 and at most 575 amino acids sequence SEQ ID N ° 33, the AMH polypeptide recognized by antibody secreted by the hybridoma is an AMH polypeptide comprising at least 100 amino acids of sequence SEQ ID N ° 32 and at most 270 amino acid sequence SEQ ID nO: 39, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 146 amino acids of sequence SEQ ID N ° 44 and at most 270 acids amino sequence SEQ ID NO: 42.

In particular :

- the mammal is human, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH selected from the polypeptide sequence SEQ ID NO 2 to 10, the polypeptide recognized by AMH the antibodies secreted by the hybridoma is a polypeptide AMH selected from the polypeptide sequence SEQ ID nO: l and SEQ ID nO 8 to 10, and the polypeptide of AMH (a) not recognized

by the antibodies secreted by the hybridoma is a polypeptide AMH selected from the polypeptide sequence SEQ ID NO: 1 to 13, or

- the mammal is a horse, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, selected from polypeptides of SEQ ID NO: sequence 15 to 20, the AMH polypeptide recognized by antibodies secreted by the hybridoma is a polypeptide AMH selected from the polypeptide sequence SEQ ID nO: 14 and SEQ ID nO: 19 and 20, and the polypeptide of AMH (a) not recognized by the antibodies secreted by hybridomas is a polypeptide AMH selected from the polypeptide sequence SEQ ID No. 21 and 22, or

- the mammal is a dog, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH selected from the polypeptide sequence SEQ ID No. 24 to 29, the AMH polypeptide recognized by antibodies secreted by the hybridoma is a polypeptide AMH selected from the polypeptide sequence SEQ ID nO: 23 and SEQ ID No. 28 and 29, and the polypeptide of AMH (a) not recognized by the antibodies secreted by hybridomas is a polypeptide AMH selected from the polypeptide sequence SEQ ID NO: 30 and 31, or

- the mammal is a bovine, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH selected from the polypeptide sequence SEQ ID No. 33 to 41, the AMH polypeptide recognized by antibodies secreted by the hybridoma is a polypeptide AMH selected from the polypeptide sequence SEQ ID No. 32 and SEQ ID nO 39 to 41, and the polypeptide of AMH (a) not recognized by the antibodies secreted by hybridomas is a polypeptide AMH selected from the polypeptide sequence SEQ ID NO: 42-44.

The antibodies produced by the method of the invention are novel and may be produced by another process, while they keep the same characteristics. Also, another object of the invention relates to antibodies or fragments of mammalian monoclonal anti-AMH antibody recognizing an AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID No. 1 or sequence having at least 75 % identity with the sequence SEQ ID nO: l and at most 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID nO: 8, but recognizes or (a) a polypeptide AMH

comprising at least the 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID NO: ll or sequence having at least 75% identity with the sequence SEQ ID No. 11 or any linear epitope positioned in the sequence SEQ ID N ° 1 or a sequence having at least 75% identity with the sequence SEQ ID No. 1.

For monoclonal antibody fragment is any portion of the antibody that has the same immunological recognition features as the antibody from which it is derived, in this case it is able to recognize the middle part of the pro region of SEP and also whole ΓΑΗΜ. Exemplary fragments include Fab, Fab ', F (ab') 2 and scFv (single chain variable fragment), dsFv (Double-stranded fragment variable). These functional fragments can in particular be obtained by genetic engineering or by specific proteolytic digestion and purification.

The characteristics and definitions described above for the method of the invention also apply to the antibodies of the invention.

Thus, antibodies of the invention may perform one or more of the following conditions:

- the polypeptide AMH (a) not recognized is a polypeptide of the sequences chosen from: SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 13 or sequences having at least

75%) identity to these sequences,

- sequences having at least 75% identity with the sequences SEQ ID No. 11, SEQ ID NO: 12, and SEQ ID NO: 13 are selected from the sequences: SEQ ID NO: 21, SEQ ID No. 22, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID N ° 44,

- linear epitopes (b) not recognized by the antibodies have the sequences SEQ ID NO: 45 to SEQ ID NO: 56.

In particular,

- the mammal is human, the AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID NO: l and at most 255 amino acids of sequence SEQ ID NO: 8, and the polypeptide of AMH (a) no

recognized is a polypeptide AMH comprising at least the 131 amino acids of sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID NO: 1, or

- the mammal is a horse, the AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID N ° 14 and at most 265 amino acids of sequence SEQ ID N ° 19, and polypeptide AMH (a) not recognized is a polypeptide AMH comprising at least the 144 amino acids of sequence SEQ ID N ° 22 and at most 166 amino acids of sequence SEQ ID No. 21, or

- the mammal is a dog, AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID N ° 23 and at most 264 amino acids of sequence SEQ ID N ° 28, and polypeptide AMH (a) not recognized is a polypeptide AMH comprising at least the 144 amino acids of sequence SEQ ID N ° 31 and at most 165 amino acids of sequence SEQ ID No. 30, or

- the mammal is a bovine, the AMH recognized polypeptide is a polypeptide AMH comprising at least the 100 amino acids of sequence SEQ ID N ° 32 and at most 270 amino acids of sequence SEQ ID N ° 39, and polypeptide AMH (a) not recognized is a polypeptide AMH comprising at least the 146 amino acids of sequence SEQ ID N ° 44 and at most 270 amino acids of sequence SEQ ID N ° 42.

Anti-AMH antibody of the invention are particularly useful for the AMH assay in a biological sample likely to contain ΓΑΜΗ. They can be used as such, in whole form or fragment, and / or in the form conjugated with a label capable of emitting a detectable signal for displaying an immunological reaction after binding with the sample ΓΑΜΗ , which constitutes another subject of the invention.

By label capable of emitting a detectable signal for displaying an immunological reaction, is meant any molecule containing a reactive group with a group of the antibody or antibody fragment directly without chemical modification, or after chemical modification to include such a group, which molecule is capable of directly or indirectly generating a detectable signal that will be used to give the desired dosage.

The bond between the label and the antibody or fragment of antibody of the invention can be performed by any method known to the skilled person, in particular by coupling.

Examples of markers include:

• enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, the enzyme glucose-6-phosphate,

• chromophores such as fluorescent, luminescent dyes,

• radioactive molecules such as 32P, 35S or 1251

• fluorescent molecules such as Alexa or phycocyanins and

• electrochemiluminescent salts such as derivatives organo -métalliques based acridinium or ruthenium.

Examples of Indirect detection systems comprise, for example ligands capable of reacting with an antiligand. The ligand corresponds to the marker to form, with the antibody or antibody fragment conjugate.

Couples ligand / anti- ligand are well known to those skilled in the art, and include for example the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary polynucleotide .

The anti-ligand may then be detectable by direct detection labels described above or be itself be detectable by another ligand / anti-ligand, and so on.

The ΓΑΜΗ was assayed by sandwich immunoassay which is a widely known test in the art. Briefly, it consists in determining an analyte, in this case mammalian ΓΑΜΗ, by implementing two binding partners to the analyte, in this case at least one antibody or antibody fragment of the invention.

Of course, the prefix "immune" in the term "immunoassay" for example, is not to be considered in this application as indicating the strictly binding partner, other than the antibody or antibody fragment of the invention is necessarily an immunologically partner such as an antibody or an antibody fragment. Indeed, as is well known in the art, this term is widely used to also refer to tests and processes in which the binding partner is not an original partner / immunological nature, but is, for example, an analyte receptor that is desired to detect and / or quantify. The essential condition is that the binding partner in question is capable of binding to the analyte, in this case ΓΑΜΗ, preferably specifically. Thus, it is known to speak of the ELISA assay for testing with non immuno logical link partners strictly speaking, known more widely in English "ligand binding assay" that could be translated in French by "trial using the ligand binding, "while the term" immunoassay "is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and quantify it, preferably specific manner, even when the binding partner other than the antibody or antibody fragment of the invention is not nature or immunological origin in the strict sense.

Another object of the invention therefore relates to a AMH quantization process mammal by sandwich immunoassay, in a biological sample likely to contain ΓΑΜΗ, which comprises or consists of the following steps:

- contacting said biological sample with two binding partners ΓΑΜΗ at least one of said partner is an antibody, an antibody fragment or conjugate of the invention,

- detecting a signal transmitted by the binding between said binding partners ΓΑΜΗ and, if present, using a label capable of emitting a detectable signal, and

- convert the signal detected at a concentration of AMH.

The first step in the quantization process comprises or consists of contacting a biological sample with two binding partners ΓΑΜΗ at least one of said partner is an antibody, antibody fragment or conjugate of the invention .

Biological samples that may contain mammalian SEP

are samples of blood, serum, plasma, follicular fluid and semen. According to one embodiment, the biological sample is a blood sample, serum or plasma.

The binding partner other than the antibody or antibody fragment antibody of the invention, optionally as a conjugate, comprises any molecule capable of binding SEP. As an example of such a binding partner include polyclonal anti-AMH antibody, anti-AMH monoclonal antibodies, fragments of anti-AMH monoclonal antibodies, antibody analogs (molecules capable of mimicking antibodies) such as nanofitines, aptamers or the "DARPins" or any other molecule that is known to interact with ΓΑΜΗ.

Analogs nanofitines antibodies are small proteins, such as antibodies, are capable of binding to a biological target and to detect, to capture or just the target within an organization.

Analogs antibody aptamers are oligonucleotides, usually RNA or DNA, identified in libraries containing up to 1015 different sequences, by a combinatorial method for selecting in vitro called SELEX "Systematic Evolution of Ligands by Exponential Enrichment" (Ellington AD and Szostak JW., 1990). Most Aptamers are compounds Darn, due to 1ARN capacity to adopt varied and complex structures, which can create on the surface cavities of various geometries, for setting various ligands. It is of interest biochemical tools that can be used in biotechnology applications, diagnosis or treatment. Their selectivity and their ligand binding properties comparable to that of antibodies.

The antibody analogues "DARPins" for Ankyrin Designed Repeat Proteins (YL Boersma and Plutckthun A, 2011) are another class of proteins to mimic the antibodies and is able to fix with high affinity and selectivity of target proteins. They derive from the family of proteins that are ankyrins adapter proteins for fixing the integral membrane protein spectrin network / actin which is "the backbone" of the cell plasma membrane. Ankyrins structure is based on the repetition of a motif of approximately 33 amino acids and it is the same of DARPins.

Each pattern has a secondary structure helix-turn-helix ( "helix-turn-helix"). The DARPins contain at least three, preferably four to five repeats and are obtained by screening of combinatorial libraries.

The polyclonal antibodies, monoclonal antibodies and fragments of anti-AMH antibody can be prepared conventionally widely known in the art. Some antibodies are commercially available, such as in AnshLabs (US).

The Applicant has also prepared other monoclonal antibodies particularly useful in the context of ΓΑΜΗ assay using the same monoclonal antibody preparation method as that described above for the invention, except that the antibody recognition region is the C-terminal part of the pro region of SEP, i.e., to human ΓΑΜΗ, amino acids 256-451 (196 amino acids SEQ ID NO: 57), for equine SEP amino acids 266-464 (199 amino acids SEQ ID NO: 58), canine ΓΑΜΗ to amino acids 265-463 (amino acid 199 SEQ ID No. 59) and for bovine AMH amino acids 271-466 (amino acid 199 SEQ ID No. 60). The C-terminal part of the pro region of interest is a polypeptide of AMH comprising at least the 196 amino acid sequence SEQ ID No. 57 or of sequence having at least 75% identity with the sequence SEQ ID N 57. Unexpectedly, the antibodies recognizing the C-terminal part of the pro region of AMH does not need to be used in large amounts in the assay in which they are implemented, unlike antibodies of art prior.

To prepare such antibodies recognizing the C-terminal part of AMH, the immunogen used is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which must include at least the portion that the antibodies must recognize, namely at least the C-terminal part of the pro region of AMH, namely at least the 196 amino acids of sequence SEQ ID N ° 57 or of sequence having at least 75% identity with the sequence SEQ ID N 57. The immunogen also comprises at most the whole of AMH species in question or the polynucleotide encoding the full AMH of the species, i.e., in the case of human AMH, the 560 amino acid sequence of SEQ ID No. 2, the other AMH comprising at least 75% identity with SEQ ID NO: 2 sequence. A particular immunogen can be selected from the following:

- a polypeptide of AMH, or polynucleotide encoding such polypeptide devoid, in relation to the entire AMH sequence, all the amino acids located after the C-terminal part of the pro region of ΓΑΜΗ, namely a polypeptide AMH, or a polynucleotide encoding said polypeptide AMH, comprising at least the 426 amino acid sequence SEQ ID NO: 7 or sequence having at least 75% identity with the sequence SEQ ID N ° 7 and at most the 451 amino acid sequence SEQ ID NO: 5 or sequence having at least 75% identity with the sequence SEQ ID NO: 5, preferably selected from polypeptides AMH sequences SEQ ID NO: 5, SEQ ID N ° 6 and SEQ ID NO: 7 or sequences having at least 75% identity to these sequences, e.g. SEQ ID NO: 17, 18, 26, 27, 36, 37 or 38. this corresponds to ΓΑΜΗ -3, where appropriate without the signal peptide and optionally without precursor portion;

- a polypeptide of AMH, or polynucleotide encoding such polypeptide, which corresponds to total ΓΑΜΗ, with all or part of the signal peptide and optionally all or part of the precursor portion, namely which comprises at least the 535 amino acid sequence of SEQ ID NO: 4 or sequence having at least 75% identity with the sequence SEQ ID NO: 4, preferably selected from polypeptides AMH sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO 4 or sequences having at least 75%) identity to these sequences, e.g. SEQ ID NO: 15, 16, 24, 25, 33, 34 or 35.

The selection of hybridoma is also done by using two steps of screenings, in any order, with individual polypeptides and peptides.

The first screening, called screening 1, is to retain only the hybridomas which produce antibodies recognizing the C-terminal part of the pro region, as described above. To do this, two methods are possible:

Method 1: First is selected hybridomas secreting antibodies recognizing SEP-3, i.e., to human SEP-3, amino acids 1-451 (SEQ ID NO: 5), where appropriate without all or part of signal peptide and part or all of the precursor portion, or any sequence having at least 75% identity with this

sequence. According to one embodiment, the AMH-3 polypeptide recognized by the antibody is a sequence polypeptide selected from: SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID No. 7 and sequences having at least 75% identity with these sequences. Then spreads the hybridomas which produce antibodies recognizing the part of ΓΑΜΗ located before the C-terminal part of the pro region of ΓΑΜΗ, namely for human SEP, amino acids 1-255 (SEQ ID NO: 8 - AMH- 2), where appropriate without all or part of the signal peptide and part or all of the precursor portion, and any sequence having at least 75% identity with this sequence. This polypeptide is referred AMH AMH polypeptide (a) not recognized by the antibodies prepared according to the invention. According to one embodiment, the polypeptide of AMH (a) not recognized by the antibody is a polypeptide sequence selected from: SEQ ID No. 8, SEQ ID No. 9 and SEQ ID NO: 10 and sequences having at least 75% identity with these sequences.

Method 2: selecting the hybridomas secreting antibodies recognizing the C-terminal part of the pro region of SEP, i.e., to human ΓΑΜΗ, amino acids 256-451 (SEQ ID NO: 57), or any sequence having at least 75%) identity with this sequence.

The second screening, called screening 2, consists of separating the hybridomas which produce antibodies which recognize linear epitopes (b) in the C-terminal portion of the pro region of ΓΑΜΗ, namely in SEQ ID NO: 57 or sequence having 75% with the sequence SEQ ID N ° 57.

The following table (Table 4) present different linear epitopes (b) determined in the C-terminal part of the pro region of ΓΑΜΗ, useful for preparing such antibodies, giving SEQ ID according to the mammalian species.

Table 4

Anti-AMH antibody thus prepared have the following characteristics: they recognize a polypeptide of AMH comprising at least the 196 amino acid sequence SEQ ID No. 57 or of sequence having at least 75% identity with the sequence SEQ ID N 57, they do not recognize (a) a polypeptide AMH comprising at least the 230 amino acid sequence SEQ ID nO: 10, or sequence having at least 75%> identity with the sequence SEQ ID No. 10 and at most 255 amino acids of sequence SEQ ID No. 8, or sequence having at least 75%> identity with the sequence SEQ ID No. 8, or (b) a linear epitope located in the sequence SEQ ID N 57 or a sequence having at least 75%> identity with the sequence SEQ ID N ° 57, for example SEQ ID NO: 61-85.

All features and definitions described above for the antibody preparation method of the invention and the antibodies of the invention also apply here.

One of the two partners can be coupled to a label to form a conjugate or a plotter. The other binding partner can be captured on a solid support, and directly or indirectly. This is known as capture partner for the latter and detection partner for the first.

Couples ΓΑΜΗ binding partners used in AMH quantification method of the invention may recognize either the pro region and the mature region of ΓΑΜΗ or only the pro region, especially when used with the antibodies the invention, an anti-AMH antibody recognizing the C-terminal part of the pro region. Thus, in a particular embodiment, the second binding partner to ΓΑΜΗ used in the quantization method is an antibody recognizing a polypeptide of AMH comprising at least the 196 amino acid sequence SEQ ID No. 57 or of sequence having at least 75% identity with the sequence SEQ ID N ° 57, but not recognizing either (a) a polypeptide AMH comprising at least the 230 amino acid sequence SEQ ID nO: 10, or sequence having at least 75% identity with the sequence SEQ ID N ° 10 and at most 255 amino acids of sequence SEQ ID No. 8, or sequence having at least 75% identity with SEQ ID No 8, or (b) a linear epitope located in the SEQ ID No 57 or a sequence having at least 75% identity with the sequence SEQ ID N ° 57, the latter being as a fragment and / or conjugated with a label capable of generate the transmit a detectable signal for viewing a reactio n immunological between said conjugate and ΓΑΜΗ a biological sample.

Contacting the biological sample with both ΓΑΜΗ binding partners may be in one stage or in two stages, as is widely known to those skilled in the art. Briefly, an immunoassay in a step comprises the presence of the sample to be tested simultaneously with the two binding partners, including antibodies, antibody fragments or conjugates of the invention as defined above, then that a two-step immunoassay comprises the presence of the test sample on the one hand with the first binding partner and the analyte-first binding partner complex thus formed is brought into the second binding partner, the one of the two binding partners is of course an antibody, antibody fragment or conjugate of the invention as defined above.

The method may also comprise other steps known to those skilled in the art, such as washing steps and incubation steps.

The second step of the process of quantization of the invention comprises or consists of the detection of a signal transmitted by the binding between said binding partners ΓΑΜΗ and, if present, using a marker capable of generating the issue of a detectable signal as defined above. This marker may be conjugated to one of said ΓΑΜΗ binding partners.

Depending on the type of labeling used, those skilled in the art will add reagents for visualizing the labeling, or the emission of the detectable signal by any suitable type of measuring device, such as a spectrophotometer, a spectrofluorimeter, a densitometer , luminometer or a high definition camera.

The last step of AMH quantization method comprises or consists of the transformation of the signal detected concentration of AMH. We also talk about rates or amounts of AMH. The general principle is that the measured signal when the immunoassay is proportional to the amount of AMH in the biological sample.

This signal processing step detected concentration of AMH is widely known to those skilled in the art. It consists of using a predetermined mathematical model from a standard range. This standard range is previously obtained in a known manner. Briefly, obtaining a calibration range is to measure the signal generated by the quantities or increasing concentrations and known to the target analyte (AMH), to plot the curve giving the signal as a function of the rate of AMH and to find a mathematical model that represents the most accurate way possible this relationship. The mathematical model is used to determine the amounts, securities or unknown concentrations of AMH contained in the biological sample to be tested.

Determining the concentration of AMH can be used at several levels, particularly in the context of fertility in women, to estimate ovarian reserve in women, for example in those who should receive extensive treatment that could destroy follicles, but also in boys before puberty, for example in case of complaints related to sexual differentiation.

Thus, another object of the invention relates to the use of antibodies, antibody fragments or conjugates of the invention, whether or not prepared from the process of the invention, or the method for quantifying the invention as an aid:

- to diagnose disorders related to ovarian dysfunction in women of reproductive age, or

- the evaluation of ovarian follicular reserve in girls over 12 years and women, or

- evaluation of disorders of sexual differentiation in boys before puberty.

To implement the methods of the invention, used in particular by the uses described above, the antibodies, fragments or conjugates of the invention may be contained in kits.

Also, another object of the invention concerns kits comprising an antibody or antibody fragment as defined above or prepared and / or a conjugate as defined above.

Again, features and definitions previously disclosed in the context of antibodies and methods of the invention apply to the kits of the invention.

According to a particular embodiment, the kits comprise also contain at least one positive control. The positive control comprises a compound capable of binding to binding partners implemented in use of the kit, the compound is present at a predetermined rate.

As non-limiting examples of such compounds include total ΓΑΜΗ sequence SEQ ID NO: 2 or sequence having at least 75% identity with SEQ ID NO: 2 sequence, optionally with part or all the signal peptide and optionally all or part of the precursor part.

The kits may also contain all the compounds for the detection of the reaction between the binding partners and ΓΑΜΗ such as wash buffers or reagents for visualizing a marking or the emission of a detectable signal .

The invention will be better understood by the following examples which are given for illustrative and not limitative, and with the aid of Figures 1 to 4, wherein:

Figure 1 is a schematic representation of the structure of human AMH protein and protein constructs AMH-1, AMH-2 and human AMH-3 derived therefrom.

Figure 2 is a Western blot analysis of cell lysates obtained by transfection of constructs, AMH-1, AMH-2, 3-AMH and complete AMH in HEK293T cells after separation by electrophoresis on a Bis-Tris gel 4- 12%. Wells 1, 2: lysate negative control (cells transfected with an empty plasmid); wells 3, 4 lysate AMH-1; wells 5, 12 standard weight

molecular; Well 6, 7: lysate AMH-2; well 8, 9 AMH-3 lysate; wells 10, 11: whole lysate AMH. Wells 1, 3, 6, 8 and 10 correspond to the condition A: the deposited samples were heated and reduced. Wells 2, 4, 7, 9 and 11 correspond to the condition B: the deposited samples were heated but not reduced. FIG 2A is a photograph of membrane tested with an anti-actin antibody, which serves to control than equivalent amounts of total protein were deposited in each well. Figure 2B is a photograph of membrane tested with an anti-histidine antibody (Qiagen). Bands that correspond to the expression of AMH constructions products have been framed.

Figure 3 is a Western blot analysis of the AMH protein-2 and AMH-3 purified from transfections supernatants of the gene constructs in HEK293T cells after separation by electrophoresis on a Bis-Tris 4-12% gel. Well 1: negative control (cells transfected with an empty plasmid); well 2: AMH-2; lane 3: AMH-3; Well 5: molecular weight standard. The deposited samples were heated but not reduced. Five identical membranes (3A to 3E) were prepared and each was tested by an anti-AMH antibody or an anti-histidine antibody (Qiagen).

Figure 4 is an SDS-PAGE analysis of the recombinant AMH protein CHO-AMH5 3F1 purified by affinity chromatography. The SDS-PAGE gel was stained with silver nitrate to visualize total proteins. Lane 1: molecular weight standard; lane 2: recombinant AMH heated but not reduced; lane 3: heated and reduced recombinant AMH.

EXAMPLES

Example 1: Cloning of DNA fragments corresponding to the entire sequence of human ΓΑΜΗ or truncated sequences, and transiently transfected into HEK 293T cell

1.1. constructs

The entire AMH sequence expressed is that of human SEP which comprises 560 amino acids (SEQ ID NO: 2 corresponding to No. P03971 accession database Uniprot KB), this construct is called AMH-560. Include the signal peptide (amino acids 1-18) and a precursor part (amino acids 19-25) which are cleaved during post-translational processing of the protein.

Three other protein constructs called AMH-1, AMH-2 and AMH-3 respectively corresponding to the sequences in amino acids 1-156 (SEQ ID NO: ll), 1-255 (SEQ ID NO: 8) and 1-451 (SEQ ID No. 5) of AMH were performed. A poly histidine tag (His-8) was added C-terminal side of these 4 constructions to facilitate purification (Tag not added in the sequence listing).

The DNA fragments corresponding to the AMH-1 constructs, AMH-2 and AMH-3 whole AMH, whose sequences are given in Table 5 below were obtained in the form of synthetic genes from the company GeneArt® (Life Technologies). Each DNA fragment (AMH-1, AMH-2, 3-AMH and complete AMH) was cloned between the Eco RI and Not I sites in the pCMV6-XL5 vector under the control of the CMV promoter. The plasmids were verified by sequencing in the inserts to ensure they do not contain errors.

Table 5

Name polynucleotide corresponding DNA fragment

d'AMH SEQ IDN°X-8HIS-codon stop

AMH-1 SEQ I D N ° 86 SEQ I D N ° 86-CACCACCATCATCACCATCACCAC-TGA

AMH-2 SEQ I D N ° 87 SEQ I D N ° 87 -CACCACCATCATCACCATCACCAC-TGA

AMH-3 SEQ I D N ° 88 SEQ I D N ° 88 -CACCACCATCATCACCATCACCAC-TGA

AMH entière SEQ IDN ° 8 9 SEQ IDN ° 8 9-CACCACCATCATCACCATCACCAC-TGA

1.2. Transient transfection in HEK293T

Culture. The HEK-293T / 17 SF (ACS-4500 ™ ATCC) were grown without serum in HEK More SFM medium (ATCC # 8006386597) enriched with glutamine (GIBCO # 250030.24), according to the manufacturer's instructions. The cells were cultured in F75 culture flasks and maintained in an incubator at 37 ° C with 5% C02 prior to transfection.

Transfection. SF HEK293T cells (10 6 cells) were transfected by nucleofection using the Amaxa Nucleofector device (Lonza), using the protocol supplied with the kit Amaxa Nucleofector Kit V cell line (# VCA-1003) and using 5 micrograms of DNA for 1 million cells per transfection. Briefly, one million cells were centrifuged at 200g for 10 minutes to harvest them. The cell pellet is then resuspended in 100 μΐ V solution (provided in the kit). 5 micrograms of plasmid was added to the cell suspension. The whole is mixed gently and transferred to a Amaxa bowl (also included in the kit). The cuvette is inserted into the Nucleofector set to Q-001 program AMAXA nucleofection and the apparatus is then activated. The sample is then immediately transferred to a warm environment to a cell culture 6-well plate will be incubated at 37 ° C, 5% C0 2 . The SF HEK293T cells are cultured for 48 hours.

Lysis and harvest. 48 hours post-transfection the supernatants were collected and frozen at -80 ° C after adding protease inhibitors (Complete ™, EDTA-free protease inhibitor cocktail tablets from Roche). Cell pellets transfected (6 X 10 6 cells / pellet) are taken up in 1.8 mL of lysis buffer phosphate 5.5 mM, NaCl 130 mM, Triton X-100 0.5% Benzonase Nuclease 5U / mL (Novagen ), MgCl 2 0.48 g / L and protease inhibitors (complete ™, EDTA-free protease inhibitor cocktail tablets, Roche Cat. No. 045-6642, 1 tablet / 50 mL, pH 7.4). The cell lysate was then placed on ice 30 minutes and then centrifuged for 15 minutes at 13,000 g, 4 ° C. The supernatants contain the AMH-1 proteins, AMH-2 and AMH-3 whole AMH, and stored at -80 ° C.

1.3. Analysis of protein expression by Western blot

A first characterization of the expression products obtained in step 1.2. was performed by SDS-PAGE analysis on a gel NuPAGE® Bis-Tris 4-12% NuPAGE ® in MES SDS buffer (Life Technologies). Before loading on the gel (14 μί / υΐίβ) transfection lysates and supernatants were diluted in the buffer NuPAGE ® LDS Sample Buffer 4X (Life Technologies) (3/1, v / v) and underwent various treatments. The reduction is done by adding 55 mM final dithiothreitol (DTT). Heating is 5 min at 75 ° C.

Condition A: HEATED and REDUCED (with DTT)

Condition B: HEATED NOT REDUCED (without DTT)

After migration of the gel, the transfer of the proteins separated by electrophoresis is performed on a nitrocellulose membrane 0.45 μιη at 350 mA constant for 50 minutes in Tris-glycine buffer IX containing 20% ​​methanol. The passivation of the membrane is carried out in the presence of 3% BSA (bovine serum albumin) in 5.5 mM phosphate buffer, 130 mM NaCl overnight at + 2/8 ° C. After passivation, a monoclonal antibody of mouse anti-histidine (Qiagen, Cat. No. 34660) is diluted at 1/2000 in phosphate buffer 5.5 mM, NaCl 130 mM containing 0.05%> Tween 20, then 10 mL of this dilution are incubated with the membrane for lh at + 18/25 ° C. A second membrane prepared in exactly the same way is incubated at the same time and under the same conditions with a mouse monoclonal anti-actin (clone AC-15, Life Technologies, Cat. No. AM4302) instead of anti-histidine antibody. This membrane is used to verify that in each well, comparable amounts of total protein were subjected to analysis (control equivalent load).

After rinsing the membrane to remove unbound antibody (5 washes for 5 min in buffer phosphate 5.5 mM, NaCl 130 mM, Tween 20 0.05%>), they were incubated for lh with a secondary antibody anti mouse conjugated to horseradish peroxidase (Jackson Immunoresearch, Cat. No. 115-036-003) diluted 1:20 000 in PBS IX, Tween 20 0.2%. After five 5 minute washes in PBS IX, Tween 20 0.2%, the visualization is carried out by incubating the membranes in a Clarity Western Blotting Substrate solution (Biorad, Cat. No. 170-5061), stirring for 5 minutes before an acquisition in chemiluminescence (ChemiDoc XRS, Biorad).

The results are shown in Figure 2. On the membrane revealed with anti-actin antibody (Figure 2A), a single band is observed per well and the intensities of these bands are equivalent, except for AMH-1 well (3 and 4), slightly less intense. This indicator is therefore validated. On the membrane revealed with anti-histidine antibody (Figure 2B), we observe bands on in the negative control wells. However, they contain a lysate of the transfected cells to white with no plasmid. This is a non-specific reactivity of the anti-histidine antibodies with certain proteins of the lysate which are found in all wells. In addition to these non-specific bands, the AMH-1 well, AMH-2 and AMH-3 whole AMH contain specific bands, that is to say not present in the negative control wells. These specific bands are framed in Figure 2B. Their apparent molecular weights for the heated and reduced condition are about 20, 30, 55 and 65 kDa respectively for AMH-1 constructs, AMH-2 and AMH-3 whole AMH (lane 3, 6, 8, 10). The apparent molecular weights for the heated but not reduced conditions, are about 36, 50, 100 and 120 kDa respectively, for AMH-1 constructs, AMH-2 and AMH-3 whole AMH (well 4, 7, 9 , 11). These estimated molecular masses are compatible with dimerization of AMH constructions in unreduced condition. It is important to note that the dimerization also occurs in the absence of the mature region and despite the significant C-terminal truncation random done in AMH AMH-1 and-2 construction.

In conclusion, this Western blot analysis shows that the plasmid constructions AMH-1, AMH-2, 3-AMH and AMH allow full well to express proteins containing a histidine tag and whose apparent molecular masses correspond to the expected molecular masses of by the nucleotide sequences.

Example 2: Expression Stable the AMH protein in CHO cells and purification

Obtaining a stable clone of CHO cells (Chinese Hamster Ovary) recombinant expressing SEP whole was performed using the cGPS Chosa CEMAX Cellectis kit (Cat. No. 0011-05 and Chosa-Chosa-0011-10 ) and following the associated protocol. This kit allows the targeted intra-chromosomal integration of a gene

exogenous CHO cell. It is composed of the cell line CHO-cGPS Sa (adherent cells), an integration vector in which the gene of interest is cloned, a vector constitutively expressing the meganuclease I-Sce I and the kit "TransMessenger ™ Transfection Reagent "(Qiagen Cat. No. 301525). The cell line CHO-cGPS The GM has the distinction of containing in its genome a particular site, large size and unique, recognized by the I-Sce I which has an endonuclease activity.

2.1. Cloning

A synthetic gene coding for the entire human AMH protein (aa 1-560) associated with a tag of 8 histidines in the C-terminal has been ordered from Geneart society. The gene is optimized for expression in CHO host. This AMH gene was cloned into the integration vector pIM.LP2.Zeo marketed by Cellectis between the Eco RI sites and Not 1 under the control of CMV promoter. The integration vector pIM.LP2.Zeo has the distinction of holding two regions homologous to the region upstream and downstream of the unique site recognized by the I-Sce I in the CHO genome. These two particular regions flank the multi insertion site into which the gene encoding ΓΑΜΗ has been inserted. In addition, this integration brings plasmid 2 selective advantage to the transfected CHO cells. The first is the gene for resistance to zeocin, controlled by the CMV promoter. The second is the resistance gene to neomycin controlled by the SV40 promoter. This gene also permits the resistance to geneticin (G418) antibiotic close to neomycin.

2.2. Transfection

One day prior to transfection (Jl), 2 x 10 5 adherent cells cGPS CHO-His CEMAX are seeded per petri dish 10 cm in F-12K medium supplemented with 2 mM L-glutamine, penicillin (100 IU / ml) streptomycin (100 mcg / ml), amphotericin B (fungizone) (0.25 g / ml) and 10% fetal calf serum.

On the day of transfection (J), 1 .mu.g of the integration vector (pIM.LP2.Zeo) containing the AMH gene and 1 .mu.g of mRNA meganuclease are diluted in buffer

EC-R (available in kit "TransMessenger ™ Transfection reagent"). 4 μΐ of Enhancer reagent are then added [nucleic acid ratio (mg) / Enhancer (ml) = ½]. The total reaction volume must be 100 μΐ. The solution is then incubated for 5 minutes at room temperature. Then 16 μΐ reagent TransMessenger ™ are added and the whole is incubated for 10 minutes at + 18/25 ° C before being deposited on the 2 x l0 5 cells whose culture medium has been previously replaced with 900 μΐ of F-12K medium without serum and without antibiotics.

2.3 Clonal selection and characterization of clones

The recombinant clones AMH were selected based on their resistance to zeocin and geneticin (G418).

24h after transfection (J + l), the culture medium was replaced with 10 ml of complete medium supplemented with 0.6 mg / ml G418 and then from the 6 th day (D + 6), the culture medium is regularly replaced with fresh medium supplemented with 0.6 mg / ml G418 and 0.4 mg / ml zeocin. 15 days after transfection (J + 15), the cells are isolated by limiting dilution in 96-well plate. After this cell cloning, clones were amplified by cultural and tested:

- PCR by driving the DNA génomique extrait des cellules CHO Blingee des des amorces spécifiques extrémités 5 'and 3' of the gene AMH,

- by Western blotting to check the expression of the protein of AMH.

Among the positive clones in both PCR and Western blot, the clone AMH5 3F 1 was selected. To clone the PCR product obtained by amplification of the AMH gene was sequenced in its entirety to ensure the integrity of the gene and make sure there was no mutation introduced in the selection.

2.4. Recombinant ΓΑΜΗ of expression from the stable clone CHO-AMH5 3F 1 clone AMH5 3F 1 is cultured in a culture dish of 225 cm 2 at a rate of 9 × 10 6 cells in 60 ml of culture medium Excell 302 (Sigma Cat. No. 4324C) supplemented with antibiotics and antifungals 1% v / v (Gibco Cat. No. 15240), 12 mM L-glutamine (Gibco Cat. No. 25030), glucose 6 g / L (Sigma Cat. No. G8769 ), 0.4 mM iron citrate (Sigma Cat. No. F6129), hypoxanthine-thymidine 2% (Gibco Cat.

No. 41065), 1% glycerol, pepstatin A 1 mg / L (Sigma Cat. No. P4265). The culture dishes were incubated in an oven at 37 ° C under an atmosphere of 7.5% C0 2 . After 4 days of culture (amplification), the cell suspension (approximately 66 × 10 6 cells) is reseeded in 7 F225 culture dishes using the same protocol. Supernatants from these cultures were harvested and centrifuged at 5000 g and frozen at -25 ° C until purification.

2.5. Purification of the recombinant AMH protein CHO-3F AMH5 1

The previously harvested culture supernatants were thawed and pooled. A volume of 5 liters of supernatant is then filtered through 0.8 μιη membrane (Nalgene, VWR Cat. No. 7345084) and 0.22 μιη membrane (Nalgene EPAS Rapid Flow). The sample is then concentrated 20 times on hollow fiber having a cutoff of 30 kDa (GE Cat. No. 564110-18). The retentate of about 100 ml is then diafiltered against 5 times its volume in potassium phosphate buffer and sodium 50 mM pH 7.8, 100 mM NaCl, 1 mM EDTA, azide 0.9 g / L. Two pellets of protease inhibitors Complete EDTA free (Roche Cat. No. 11873580001) are added. This retentate is purified by affinity chromatography on Sepharose Hi-Trap NHS column coupled to an anti-AMH antibody (polyclonal or monoclonal) at 10 mg of antibody per ml of gel according to the supplier protocol (GE Cat. No. 17-0716-01). The affinity column is equilibrated in diafiltration buffer and then 50 ml of retentate are injected onto the column at a rate of 0.5 ml / min. Then washing is carried out in buffer 50 mM Tris HCl pH 7.9, 100 mM NaCl, ImM EDTA. Elution of the AMH protein is then achieved by a Glycine-HCl buffer 0.1 M pH 2.9 at a rate of 0.5 ml / min. The collected elution fractions are immediately neutralized to pH 7-8, supplemented with protease inhibitors, pooled and stored at -80 ° C in a storage buffer NaHC0 3 0.2M, 0.5M NaCl pH 7.5 and 20% ethanol. 4 shows the photograph of the membrane following an analysis by SDS-PAGE of the recombinant AMH protein CHO-3F AMH5 1 according to the protocol of Example 1.3. In place of the Western blot staining with silver nitrate total protein was performed. The purified protein is very pure.

Example 3: Anti-AMH antibody Preparation of immunizing mice by invention

3.1. the immunogenic

Plasmids entire AMH and AMH-3 obtained in Example 1 were amplified by growth in E. coli bacteria and purified using the EndoFree Plasmid Mega Kit from Qiagen kit (Cat. No. 12381) or equivalent kit. For the preparation of Gene Gun cartridge (Bio-Rad), 2 ug of plasmid DNA were precipitated on 0.22 mg of gold beads of 1 μιη diameter in the presence of CaCl 2 and spermidine, as directed by the maker. The thus prepared beads may be stored at + 2/8 ° C in the dark, in the presence of a moisture absorber (desiccant sachet).

Protein recombinant AMH CHO-3F AMH5 1 obtained in Example 2 was mixed volume for volume with Freund's adjuvant (Sigma), prepared as water-in-oil emulsion of which it is known that has good power adjuvant. This preparation was performed extemporaneously before each injection.

3.2. immunizations

The immunization experiments were carried out in BALB / c (H-2 d ) mice six to eight weeks at the time of the first immunization. Different protocols are implemented:

• 4 doses of 4 mg per AMH entire DNA injection at 0, 2, 4 and 6 weeks

• 4 doses of 4 mg per injection of DNA AMH-2 at 0, 2, 4 and 6 weeks

• 4 doses of 4 mg per injection of DNA AMH-3 at 0, 2, 4 and 6 weeks

• 3 doses of 10 mg per injection of AMH protein at 0, 2, and 4 weeks.

For the DNA immunizations, the mice were razed to the abdomen. The Helios Gene Gun Delivery System (Bio-Rad) was used at a pressure of 2750 kPa for injecting the gold beads coated with DNA into mouse skin. For protein immunizations, the injection was performed by subcutaneously.

To monitor the development of antibodies, regularly is performed on mice blood samples. The presence of anti-AMH antibodies in these sera is tested by performing a 96-well ELISA microplate. The recombinant protein AMH CHO

AMH5 3F1 is used for capture (1
;. After saturation is reacted with this antigen different dilutions of sera to be tested (incubation at 37 ° C for lh) Anti-AMH antibody present in the serum are revealed by a goat antibody anti-mouse IgG conjugated to alkaline phosphatase AffmiPure (H + L, Jacskon Immunoresearch, Cat no. 115-055-146), which binds the desired antibodies (0.1 ug / well). It thus identifies among mice immunized, those who developed anti-AMH antibody.

From 50 to 70 days after the first injections, the mice who developed anti-AMH antibody response were restimulated by intravenous injection of 100 ug protein of AMH.

3.3. Preparation of hybridomas

Three days after the last injection, the mice were sacrificed respondents; blood and spleen were collected. The splenocytes obtained from the spleen were cultured with myeloma cells Sp2 / 0-for Agl4 they fuse and become immortalized, according to the protocol described by Ko yesterday and Milstein (Kohler and Milstein, 1975, and Kohler al, 1976). After a cultivation period of 12-14 days of hybridomas supernatants were screened for the presence of anti-AMH antibody using the ELISA assay described in the previous paragraph.

3.4. Selection of hybridoma supernatants recognizing the region 157-255 or 256-451 region of human AMH protein

An anti-rabbit 6 histidines Anticops (Sigma Aldrich, Cat. No. SAB4301134 or equivalent) was diluted in IX PBS and adsorbed on a microtiter 96-well plate at a rate of 1 .mu.g / well, for incubation overnight at + 18/25 ° C. The plate is then washed 3 times in PBS-Tween 20 buffer 0.05% (PBS-T) and then passivated by incubation in a PBS-T buffer containing 10 g / L BSA and washed again 3 times in PBS-buffer T.

The lysates obtained in Example 1 and stored at -80 ° C are thawed and diluted 1/2 to 1/10 in PBS IX buffer. 100 μΐ of each of the lysates (negative control, AMH-1, AMH-2, 3-AMH and complete AMH) are distributed in several wells of the

plate and incubated 2h at 37 ° C. Then, the plate is emptied and washed 4 times in PBS-T buffer containing 300 mM NaCl. The hybridoma supernatants to be tested (100 μΐ) were then added to each of 5 different lysates and incubated lh at 37 ° C. After a further step 4 washes in PBS-T 300 mM NaCl was added the secondary antibody which is an AffmiPure anti-mouse IgG made in goat, conjugated to peroxidase (H + L, Jacskon Immunoresearch, Cat no. 115-035-166). After a new phase of 3 PBS-T washes in 300 mM NaCl, the reaction was revealed by incubating the plate 10 min at + 18/25 ° C in the presence of the substrate SureBlue TMB Microwell Peroxidase ™ (KLP, Cat. No. 52- 00-01). Reading the plate is made by measuring the OD at 450 and 630 nm. Selection of hybridoma supernatants recognizing the 157-255 region. The hybridomas selected from the results of the ELISA are those whose supernatants contain antibodies that recognize the WHA-2 lysate and do not recognize the AMH-1 lysate. Of course, the AMH-3 lysates and whole AMH are also recognized by the supernatants of the selected hybridomas. The 1B11 and 5G5 hybridomas were selected.

Selection of hybridoma supernatants recognizing the 256-451 region. The hybridomas selected from the results of the ELISA are those whose supernatants contain antibodies that recognize the AMH-3 lysate and do not recognize the WHA-2 lysate nor the AMH-1 lysate. Of course, the AMH entire lysate is also recognized by the supernatants of the selected hybridomas. The hybridomas 3H8, 4C7 and 4G10 were selected.

After selection, the selected hybridomas were cloned according to the limiting dilution technique, well known in the art to ensure clonality. It was thus possible to obtain monoclonal hybridomas secreting anti-AMH antibody following:

5G5A10 1B11B1 and that recognizing the 157-255 region of human SEP - 3H8E2, 4C7E12 4G10E12 and that recognizing the 256-451 region of human AMH.

The large scale production of monoclonal antibodies was performed by culturing the hybridoma in the bioreactor miniPERM ™, according to a protocol derived from the publication of Falkenberg (1998). The monoclonal antibodies were then purified from the culture supernatant by affinity chromatography on protein A.

Example 4: Selection of anti-AMH monoclonal antibodies recognizing the non-linear epitopes

Among the monoclonal antibodies obtained in Example 3, we determined those directed against linear epitopes in order to select those that recognize non-linear epitopes. To do this, 80 synthetic peptides covering the entire amino acid sequence of human ΓΑΜΗ were synthesized. Antibody binding to each of these peptides was tested by ELISA.

4.1. peptide synthesis

80 peptides of 16 amino acids were synthesized. Of the 16 amino acids, 12 correspond to the sequence of ΓΑΜΗ and overlap of 5 amino acids and covering the entire human ΓΑΜΗ sequence (amino acids 1-560). At the N-terminus, a biotin then SGSG sequence (spacer arm) has been added to facilitate the analysis of these peptides in ELISA. These peptides are not purified and in solution (water / acetonitrile). They were checked by LC / MS mass spectrometry. The synthesized sequences are presented in Table 6.

Table 6. Sequences of the peptides human AMH.

n° Peptide d'AMH

Positions total peptide sequence (SEQ ID NO)

MRDLPLTSLALV

1 1-12 Bio-GSG-MRDLPLTSLALV-amide

(SEQ ID N°90)

SLALVLSALGAL

2 8-19 Bio-GSG-SLALVLSALGAL-amide

(SEQ ID N°91)

ALGALLGTEALR

3 15-26 Bio-GSG-ALGALLGTEALR-amide

(SEQ ID N°92)

TEALRAEEPAVG

4 22-33 Bio-GSG-TEALRAEEPAVG-amide

(SEQ ID N°93)

EPAVGTSGLIFR

5 29-40 Bio-GSG-EPAVG SGLIFR-amide

(SEQ ID N°94)

GLIFREDLDWPP

36-47 Bio-SGSG-GLIFREDLDWPP-amide (SEQ ID N°95)

LDWPPGSPQEPL

43-54 Bio-SGSG-LDWPPGSPQEPL-amide (SEQ ID N°96)

PQEPLCLVALGG

50-61 Bio-SGSG-PQEPLCLVALGG-amide (SEQ ID N°97)

VALGGDSNGSSS

57-68 Bio-GSG-VALGGDSNGSSS-amide (SEQ ID N ° 98)

NGSSSPLRWGA

64-75 Bio-GSG-NGSSSPLRWGA-amide (SEQ ID N ° 99)

RWGALSAYEQA

71-82 Bio-SGSG-RWGALSAYEQA-amide (SEQ ID N°100)

AYEQAFLGAVQR

78-89 Bio-SGSG-AYEQAFLGAVQR-amide (SEQ ID N°101)

GAVQRARWGPRD

85-96 Bio-SGSG-GAVQRARWGPRD-amide (SEQ ID N°102)

WGPRDLATFGVC

92-103 Bio-SGSG-WGPRDLATFGVC-amide (SEQ ID N°103)

TFGVCNTGDRQA

99-110 Bio-SGSG- FGVCNTGDRQA-amide (SEQ ID N°104)

GDRQAALPSLRR

106-117 Bio-SGSG-GDRQAALPSLRR-amide (SEQ ID N°105)

PSLRRLGAWLRD

113-124 Bio-SGSG-PSLRRLGAWLRD-amide (SEQ ID N°106)

AWLRDPGGQRLV

120-131 Bio-SGSG-AWLRDPGGQRLV-amide (SEQ ID N°107)

GQRLWLHLEEV

127-138 Bio-GSG-GQRLWLHLEEV-amide (SEQ ID N ° 108)

HLEEVTWEPTPS

134-145 Bio-SGSG-HLEEVTWEPTPS-amide (SEQ ID N°109)

EPTPSLRFQEPP

141-152 Bio-SGSG-EPTPSLRFQEPP-amide (SEQ ID N°110)

FQEPPPGGAGPP

148-159 Bio-SGSG-FQEPPPGGAGPP-amide (SEQ ID N°lll)

GAGPPELALLVL

155-166 Bio-GSG-GAGPPELALLVL-amide (SEQ ID N ° 112)

ALLVLYPGPGPE

162-173 Bio-GSG-ALLVLYPGPGPE-amide (SEQ ID N ° 113)

GPGPEVTVTRAG

169-180 Bio-SGSG-GPGPEVTVTRAG-amide (SEQ ID N°114)

VTRAGLPGAQSL

176-187 Bio-SGSG-VTRAGLPGAQSL-amide (SEQ ID N°115)

GAQSLCPSRDTR

183-194 Bio-SGSG-GAQSLCPSRDTR-amide (SEQ ID N°116)

SRDTRYLVLAVD

190-201 Bio-GSG-SRDTRYLVLAVD-amide (SEQ ID N ° 117)

VLAVDRPAGAWR

197-208 Bio-SGSG-VLAVDRPAGAWR-amide (SEQ ID N°45)

AGAWRGSGLALT

204-215 Bio-GSG-AGAWRGSGLALT-amide (SEQ ID N ° 118)

GLALTLQPRGED

211-222 Bio-SGSG-GLALTLQPRGED-amide (SEQ ID N°119)

PRGEDSRLSTAR

218-229 Bio-SGSG-PRGEDSRLSTAR-amide (SEQ ID N°46)

LSTARLQALLFG

225-236 Bio-SGSG-LSTARLQALLFG-amide (SEQ ID N°120)

ALLFGDDHRCFT

232-243 Bio-SGSG-ALLFGDDHRCF -amide (SEQ ID N°121)

HRCFTRMTPALL

239-250 Bio-SGSG-HRCFTRMTPALL-amide (SEQ ID N°47)

TPALLLLPRSEP

246-257 Bio-SGSG-TPALLLLPRSEP-amide (SEQ ID N°122)

PRSEPAPLPAHG

253-264 Bio-GSG-PRSEPAPLPAHG-amide (SEQ ID N ° 123)

LPAHGQLDTVPF

260-271 Bio-GSG-LPAHGQLDTVPF-amide (SEQ ID N ° 61)

DTVPFPPPRPSA

267-278 Bio-SGSG-DTVPFPPPRPSA-amide (SEQ ID N°124)

PRPSAELEESPP

274-285 Bio-GSG-PRPSAELEESPP-amide (SEQ ID N ° 125)

EESPPSADPFLE

281-292 Bio-SGSG-EESPPSADPFLE-amide (SEQ ID N°126)

DPFLETLTRLVR

288-299 Bio-SGSG-DPFLETLTRLVR-amide (SEQ ID N°127)

TRLVRALRVPPA

295-306 Bio-SGSG-TRLVRALRVPPA-amide (SEQ ID N°128)

RVPPARASAPRL

302-313 Bio-SGSG-RVPPARASAPRL-amide (SEQ ID N°129)

SAPRLALDPDAL

309-320 Bio-GSG-SAPRLALDPDAL-amide (SEQ ID N ° 130)

DPDALAGFPQGL

316-327 Bio-SGSG-DPDALAGFPQGL-amide (SEQ ID N°131)

FPQGLVNLSDPA

323-334 Bio-GSG-FPQGLVNLSDPA-amide (SEQ ID N ° 132)

LSDPAALERLLD

330-341 Bio-SGSG-LSDPAALERLLD-amide (SEQ ID N ° 62)

ERLLDGEEPLLL

337-348 Bio-SGSG-ERLLDGEEPLLL-amide (SEQ ID N°63)

EPLLLLLRPTAA

344-355 Bio-GSG-EPLLLLLRPTAA-amide (SEQ ID N ° 64)

RPTAATTGDPAP

351-362 Bio-GSG-RPTAATTGDPAP-amide (SEQ ID N ° 133)

GDPAPLHDPTSA

358-369 Bio-SGSG-GDPAPLHDPTSA-amide (SEQ ID N°65)

DPTSAPWATALA

365-376 Bio-SGSG-DPTSAPWATALA-amide (SEQ ID N°66)

A ALARRVAAEL

372-383 Bio-GSG-ATALARRVAAEL-amide (SEQ ID N ° 134)

VAAELQAAAAEL

379-390 Bio-SGSG-VAAELQAAAAEL-amide (SEQ ID N°135)

AAAELRSLPGLP

386-397 Bio-GSG-AAAELRSLPGLP-amide (SEQ ID N ° 136)

LPGLPPATAPLL

393-404 Bio-GSG-LPGLPPATAPLL-amide (SEQ ID N ° 137)

TAPLLARLLALC

400-411 Bio-GSG-TAPLLARLLALC-amide (SEQ ID N ° 138)

LLALCPGGPGGL

407-418 Bio-GSG-LLALCPGGPGGL-amide (SEQ ID N ° 139)

GPGGLGDPLRAL

414-425 Bio-SGSG-GPGGLGDPLRAL-amide (SEQ ID N°140)

PLRALLLLKALQ

421-432 Bio-GSG-PLRALLLLKALQ-amide (SEQ ID N ° 141)

LKALQGLRVEWR

62 428-439 Bio-GSG-LKALQGLRVEWR-amide

(SEQ ID N°67)

RVEWRGRDPRGP

63 435-446 Bio-GSG-RVEWRGRDPRGP-amide

(SEQ ID N°68)

DPRGPGRAQRSA

64 442-453 Bio-GSG-DPRGPGRAQRSA-amide

(SEQ ID N°142)

AQRSAGATAADG

65 449-460 Bio-GSG-AQRSAGATAADG-amide

(SEQ ID N°143)

TAADGPCALREL

66 456-467 Bio-SGSG- AADGPCALREL-amide

(SEQ ID N°144)

ALRELSVDLRAE

67 463-474 Bio-GSG-ALRELSVDLRAE-amide

(SEQ ID N°145)

DLRAERSVLIPE

68 470-481 Bio-GSG-DLRAERSVLIPE-amide

(SEQ ID N°146)

VLIPETYQANNC

69 477-488 Bio-GSG-VLIPETYQANNC-amide

(SEQ ID N°147)

QANNCQGVCGWP

70 484-495 Bio-GSG-QANNCQGVCGWP-amide

(SEQ ID N°148)

VCGWPQSDRNPR

71 491-502 Bio-GSG-VCGWPQSDRNPR-amide

(SEQ ID N°149)

DRNPRYGNHWL

72 498-509 Bio-GSG-DRNPRYGNHWL-amide

(SEQ ID N°150)

NHWLLLKMQVR

73 505-516 Bio-GSG-NHWLLLKMQVR-amide

(SEQ ID N°151)

KMQVRGAALARP

74 512-523 Bio-GSG-KMQVRGAALARP-amide

(SEQ ID N°152)

ALARPPCCVPTA

75 519-530 Bio-GSG-ALARPPCCVPTA-amide

(SEQ ID N°153)

CVPTAYAGKLLI

76 526-537 Bio-GSG-CVPTAYAGKLLI-amide

(SEQ ID N°154)

GKLLISLSEERI

77 533-544 Bio-GSG-GKLLISLSEERI-amide

(SEQ ID N°155)

SEERISAHHVPN

78 540-551 Bio-SGSG-SEERISAHHVPN-amide

(SEQ ID N°156)

HHVPNMVATECG

79 547-558 Bio-GSG-HHVPNMVATECG-amide

(SEQ ID N°157)

VPNMVATECGCR

80 549-560 Bio-GSG-VPNMVATECGCR-amide

(SEQ ID N°158)

4.2. ELISA

The 96-well microplates are coated with streptavidin (10 μ§ / ι 1, 1 μ g / well) in PBS IX for lh at 37 ° C, then passivated in a buffer PBS-Tween 20 0.05% (PBS T) containing 10 g / 1 BSA overnight at room temperature. The passivation solution is removed, and then the biotinylated peptides are distributed (10 μ§ / ι 1 in buffer PBS IX, 1 .mu.g per well) and incubated lh at 37 ° C. After 4 washes in PBS-T, the monoclonal antibody was added to test at a concentration of 1 mcg / ml. After incubation for 37 h 30 min at 4 ° C and PBS-T washes, adding a mouse anti-IgG antibody conjugated to peroxidase. Revelation is made with TMB substrate with measuring the optical density (OD) at 450 nm.

CLAIMS
1. A method of preparing mammalian anti-antibody AMH (Anti-Mullerian hormone mammalian), characterized in that it comprises the steps of:
(I) immunizing an animal with a polypeptide of AMH or a polynucleotide encoding the polypeptide of AMH, AMH said polypeptide comprising at least 99 amino acids of SEQ ID NO: l sequence or sequence having at least 75% identity with the sequence SEQ ID No. 1, and at most 560 amino acids of sequence SEQ ID NO: 2 or sequence having at least 75% identity with the sequence SEQ ID No. 2,

(Ii) preparing hybridomas from cells of a lymphoid organ of the animal that received the immunogen,

(Iii) selecting the hybridomas secreting antibodies recognizing a AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID No. 1 or sequence having at least 75% identity with the sequence SEQ ID NO: l, and at most 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID N ° 8, but not recognizing either (a) a polypeptide comprising at least the AMH 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID No. 11 or of sequence having at least 75% identity to SEQ ID no 11, or (b) no linear epitope in SEQ ID no 1 or a sequence having at least 75% identity with the sequence SEQ ID nO: l, and

(Iv) production of antibodies.

2. A process for preparing anti-AMH antibody according to claim 1, wherein the immunogen used is a polypeptide of AMH, or a polynucleotide encoding said polypeptide AMH, which comprises at least the 230 amino acid sequence SEQ ID No. 10 or of sequence having at least 75% identity with the sequence SEQ ID N ° 10 and at most 255 amino acids of sequence SEQ ID NO 8 or sequence having at least 75% identity with the sequence SEQ ID NO: 8, of

preferably selected from polypeptides AMH sequences SEQ ID No. 8, SEQ ID No. 9 and SEQ ID NO: 10 or sequences having at least 75% identity with these sequences.

3. A process for preparing anti-AMH antibody according to claim 1, wherein the immunogen used is a polypeptide of AMH, or a polynucleotide encoding said polypeptide AMH, which comprises at least the 535 amino acid sequence SEQ ID NO: 4 or sequence having at least 75% identity with the sequence SEQ ID NO: 4, preferably selected from polypeptides AMH sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID No. 4 or sequences having at least 75% identity with these sequences.

4. A process for preparing anti-AMH antibody according to any one of claims 1 to 3, wherein the polypeptide of AMH (a) not recognized by the antibody is a polypeptide sequence selected from: SEQ ID No. 11 , SEQ ID NO: 12 and SEQ ID NO: 13 and sequences having at least 75% identity with these sequences.

5. A process for preparing anti-AMH antibody according to claim 4, wherein the sequences having at least 75% identity with the sequences SEQ ID N ° 11,

SEQ ID NO: 12, and SEQ ID NO: 13 are selected from the sequences: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID N ° 43 and SEQ ID NO: 44.

6. A process for preparing anti-AMH antibody according to any one of claims 1 to 4, wherein the linear epitopes (b) not recognized by the antibodies have the sequences SEQ ID NO: 45 to SEQ ID NO: 56.

7. A process for preparing anti-AMH antibody according to any one of claims 1 to 5, wherein

- the mammal is human, the immunogen is a polypeptide of AMH, or

the polynucleotide encoding the polypeptide of AMH, which comprises at least the 99 amino acids of sequence SEQ ID N ° 1 and at most 560 amino acids of sequence SEQ ID N ° 2, the AMH polypeptide recognized by antibody secreted by hybridomas AMH is a polypeptide comprising at least 99 amino acids of sequence SEQ ID nO: l and at most 255 amino acids of sequence SEQ ID No. 8, and the polypeptide of AMH (a) not recognized by the antibodies secreted by the hybridoma is an AMH polypeptide comprising at least 131 amino acids of sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID NO: l 1, or

- the mammal is a horse, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 99 amino acids of sequence SEQ ID N ° 14 and at most 573 amino acids of SEQ ID No. 15 sequence, the polypeptide of AMH recognized by the antibodies secreted by hybridomas is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID N ° 14 and at most 265 amino acid sequence SEQ ID nO: 19, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 144 amino acids of sequence SEQ ID N ° 22 and at most 166 acids amines of sequence SEQ ID N ° 21, or

- the mammal is a dog, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 99 amino acids of sequence SEQ ID N ° 23 and at most 572 amino acids sequence SEQ ID N ° 24, the AMH polypeptide recognized by antibody secreted by the hybridoma is an AMH polypeptide comprising at least 99 amino acids of sequence SEQ ID N ° 23 and at most 264 amino acid sequence SEQ ID nO: 28, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 144 amino acids of sequence SEQ ID N ° 31 and at most 165 acids amino sequence SEQ ID NO: 30, or

- the mammal is a bovine, the immunogen is a polypeptide of AMH, or a polynucleotide encoding the polypeptide of AMH, which comprises at least the 100 amino acids of sequence SEQ ID No. 32 and at most 575 amino acids sequence SEQ ID N ° 33, the AMH polypeptide recognized by antibody secreted by the hybridoma is an AMH polypeptide comprising at least 100 amino acids of sequence SEQ ID N ° 32 and at most 270 amino acid sequence SEQ ID nO: 39, and the polypeptide of AMH (a) not recognized by the antibody secreted by the hybridoma is an AMH polypeptide comprising at least 146 amino acids of sequence SEQ ID N ° 44 and at most 270 acids amino sequence SEQ ID NO: 42.

8. A monoclonal antibody or anti-AMH mammalian monoclonal antibody fragment recognizing a AMH polypeptide comprising at least 99 amino acids of SEQ ID NO sequence or the sequence having at least 75% identity with the sequence SEQ ID nO: l and at most 255 amino acids of sequence SEQ ID nO 8 or sequence having at least 75% identity with the sequence SEQ ID nO: 8, but recognizes neither (a) a polypeptide of AMH comprising at least the 131 amino acid sequence SEQ ID No. 13 or of sequence having at least 75% identity with the sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID NO: ll or sequence having at least 75% identity with the sequence SEQ ID No. 11 or any linear epitope positioned in the sequence SEQ ID N ° 1 or a sequence having at least 75% identity with the sequence SEQ ID nO: .

9. A monoclonal antibody or fragment of mammalian anti-AMH monoclonal antibody according to claim 8, wherein the polypeptide of AMH (a) not recognized is a polypeptide of the sequences chosen from: SEQ ID NO: 1, SEQ ID 12 and SEQ ID NO: 13 or sequences having at least 75% identity with these sequences.

10. A monoclonal antibody or anti-AMH mammalian monoclonal antibody fragment according to claim 9, for which the sequences having at least 75% identity with the sequences SEQ ID No. 11, SEQ ID NO: 12, and SEQ ID # 13 are selected from the sequences: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID No 44 .

11. A monoclonal antibody or anti-AMH mammalian monoclonal antibody fragment according to any one of claims 8 to 10 for the linear epitopes (b) not recognized by the antibodies have the sequences SEQ ID NO: 45 to

SEQ ID N°56.

12. A monoclonal antibody or anti-AMH mammalian monoclonal antibody fragment according to any one of claims 9 to 11, wherein

- the mammal is human, the AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID NO: l and at most 255 amino acids of sequence SEQ ID NO: 8, and the polypeptide of AMH (a) not recognized is a polypeptide AMH comprising at least the 131 amino acids of sequence SEQ ID N ° 13 and at most 156 amino acids of sequence SEQ ID nO: l 1, or - the mammal is horse, AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID N ° 14 and at most 265 amino acids of sequence SEQ ID NO: 19, and the polypeptide of AMH (a) not recognized is a polypeptide AMH comprising at least the 144 amino acids of sequence SEQ ID N ° 22 and at most 166 amino acids of sequence SEQ ID No. 21, or - the mammal is a dog, AMH recognized polypeptide is a polypeptide AMH comprising at least 99 amino acids of sequence SEQ ID N ° 23 and at most 264 acids amino sequence SEQ ID NO: 28, and the polypeptide of AMH (a) not recognized is a polypeptide AMH comprising at least the 144 amino acids of sequence SEQ ID N ° 31 and at most 165 amino acids of sequence SEQ ID No. 30, or - the mammal is a bovine, the AMH recognized polypeptide is a polypeptide AMH comprising at least the 100 amino acids of sequence SEQ ID N ° 32 and at most 270 amino acids of sequence SEQ ID # 39, and the polypeptide of AMH (a) not recognized is a polypeptide AMH comprising at least the 146 amino acids of sequence SEQ ID N ° 44 and at most 270 amino acids of sequence SEQ ID N ° 42.

13. A conjugate comprising (i) a monoclonal antibody or a fragment of anti-AMH monoclonal antibody as defined in any one of claims 8 to 12 or prepared by the process as defined in any one of claims 1 to 7 and (ii) a label capable of issuing a detectable signal for displaying an immunological reaction between said conjugate and ΓΑΜΗ a biological sample.

14. AMH quantization method mammal by sandwich immunoassay, in a biological sample likely to contain ΓΑΜΗ, which comprises the steps of:

- contacting said biological sample with two binding partners ΓΑΜΗ at least one of said partner is an antibody or antibody fragment such as defined in any one of claims 8 to 12 or as prepared according to the method as defined in any one of claims 1 to 7, or a conjugate as defined in claim 13,

- detecting a signal transmitted by the binding between said binding partners ΓΑΜΗ and, if present, using a label capable of emitting a detectable signal, and

- convert the signal detected at a concentration of AMH.

15. A method of quantifying AMH according to claim 14, wherein the biological sample is a blood sample, serum or plasma.

16. A method of quantifying AMH according to one of claims 14 or 15, wherein the second SEP binding partner is an antibody recognizing an AMH polypeptide comprising at least the 196 amino acid sequence SEQ ID NO: 57 or sequence having at least 75% identity with the sequence SEQ ID N ° 57, but not recognizing either (a) a polypeptide AMH comprising at least the 230 amino acid sequence SEQ ID nO: 10, or sequence having at least 75% identity with the sequence SEQ ID N ° 10 and at most 255 amino acids of sequence SEQ ID No. 8, or sequence having at least 75% identity with the sequence SEQ ID No. 8 or (b) a linear epitope located in the SEQ ID No 57 or a sequence having at least 75% identity with the sequence SEQ ID N ° 57.

17. Use of a method of quantifying AMH as defined in any one of claims 14 to 16 as an aid for the diagnosis of disorders associated with ovarian dysfunction in women of childbearing age.

18. Use of a method of quantifying AMH as defined in any one of claims 14 to 16 as an aid in the evaluation of ovarian follicular reserve among young women over 12 years and women.

19. Use of a method of quantifying AMH as defined in any one of claims 14 to 16 as an aid in the evaluation of disorders related to sexual differentiation in boys before the puberty.

20. A kit comprising an antibody or antibody fragment such as defined in any one of claims 8 to 12 or as prepared according to the method as defined in any one of claims 1 to 7, and / or a conjugate as defined in claim 13.