Method for preparing anti-AMH antibodies and uses of same
The present invention relates to a method for preparing anti-mammalian AMH antibodies comprising the steps of: (i) immunizing an animal with an AMH polypeptide or a polynucleotide encoding this AMH polypeptide, said AMH polypeptide comprising at least the 99 amino acids of sequence SEQ ID No. 1 or of a sequence having at least 75% identity with the sequence SEQ ID No. 1, and at most the 560 amino acids of sequence SEQ ID No. 2 or of a 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 having received the immunogen, (iii) selecting hybridomas secreting antibodies recognizing an AMH polypeptide comprising at least the 99 amino acids of sequence SEQ ID No. 1 or of a sequence having at least 75% identity with the sequence SEQ ID No. 1, and at most the 255 amino acids of sequence SEQ ID No. 8 or of a sequence having at least 75% identity with the sequence SEQ ID No. 8, but recognizing neither (a) an AMH polypeptide comprising at least the 131 amino acids of sequence SEQ ID No. 13 or of a sequence having at least 75% identity with the sequence SEQ ID No. 13 and at most the 156 amino acids of sequence SEQ ID No. 11 or of a sequence having at least 75% identity with the sequence SEQ ID No. 11, nor (b) any linear epitope located in the sequence SEQ ID No. 1 or a sequence having at least 75% identity with the sequence SEQ ID No. 1, and (iv) producing the antibodies. The invention also relates to the antibodies and antibody fragments and the use of same for assaying AMH, in particular in fertility.
1. A method for preparing anti-mammalian anti-Müllerian hormone (AMH) antibodies, comprising:
(i) immunizing an animal with an immunogen comprising an AMH polypeptide or a polynucleotide encoding the AMH polypeptide, the AMH polypeptide comprising a sequence selected from the group consisting of:
the human sequence of SEQ ID No. 1,
the equine sequence of SEQ ID No. 14,
the canine sequence of SEQ ID No. 23, and
the bovine sequence of SEQ ID No. 32;
(ii) preparing hybridomas from cells of a lymphoid organ of the animal immunized with the immunogen;
(iii) selecting one or more of the hybridomas that each secrete an antibody recognizing a non-linear epitope of a sequence selected from the group consisting of:
the human sequence of SEQ ID No. 1,
the equine sequence of SEQ ID No. 14,
the canine sequence of SEQ ID No. 23, and
the bovine sequence of SEQ ID No. 32,
the non-linear epitope being located at a median portion of the pro region of AMH, by contacting the antibody with an AMH polypeptide selected from the group consisting of:
the sequence of SEQ ID No. 8, or a fragment of SEQ ID No. 8 comprising SEQ ID No. 1,
the sequence of SEQ ID No. 20, or a fragment of SEQ ID No. 20 comprising SEQ ID No. 14,
the sequence of SEQ ID No. 29, or a fragment of SEQ ID No. 29 comprising SEQ ID No. 23, and
the sequence of SEQ ID No. 41, or a fragment of SEQ ID No. 41 comprising SEQ ID No. 32; and
(iv) producing anti-AMH antibodies by culturing the selected hybridomas.
2. The method as claimed in claim 1 , wherein the antibody does not recognize linear epitopes of SEQ ID No. 45 to SEQ ID No. 56.
3. The method as claimed in claim 1 , wherein:
the AMH polypeptide is the SEQ ID No. 8, or a fragment of SEQ ID No. 8 comprising SEQ ID No. 1, and
the one or more selected hybridomas each secrete an antibody recognizing a non-linear epitope of the human sequence of SEQ ID No. 1.
4. The method as claimed in claim 3 , wherein the AMH polypeptide comprises SEQ ID No. 10.
5. The method as claimed in claim 3 , wherein the antibody does not recognize any of SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13.
6. The method as claimed in claim 1 , wherein:
the AMH polypeptide is the SEQ ID No. 20, or a fragment of SEQ ID No. 20 comprising SEQ ID No. 14, and
the one or more selected hybridomas each secrete an antibody recognizing a non-linear epitope of the equine sequence of SEQ ID No. 14.
7. The method as claimed in claim 6 , wherein the antibody does not recognize any of SEQ ID No. 21 and SEQ ID No. 22.
8. The method as claimed in claim 1 , wherein:
the AMH polypeptide is the SEQ ID No. 29, or a fragment of SEQ ID No. 29 comprising SEQ ID No. 23, and
the one or more selected hybridomas each secrete an antibody recognizing a non-linear epitope of the canine sequence of SEQ ID No. 23.
9. The method as claimed in claim 8 , wherein the antibody does not recognize any of SEQ ID No. 30 and SEQ ID No. 31.
10. The method as claimed in claim 1 , wherein:
the AMH polypeptide is the SEQ ID No. 41, or a fragment of SEQ ID No. 41 comprising SEQ ID No. 32, and
the one or more selected hybridomas each secrete an antibody recognizing a non-linear epitope of the bovine sequence of SEQ ID No. 32.
11. The method as claimed in claim 10 , wherein the antibody does not recognize any of SEQ ID No. 42, SEQ ID No. 43, and SEQ ID No. 44.