Method for preparing anti-AMH antibodies and uses of same
11225518 · 2022-01-18
Assignee
Inventors
- Yasemin Ataman-Onal (Reyrieux, FR)
- Sylvie Cheucle (La Tour de Salvagny, FR)
- Maxime Combe (Lyons, FR)
- Soizic Daniel (Trevoux, FR)
- Sophie Ottone (Sain-Bel, FR)
Cpc classification
G01N33/74
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
C07K16/26
CHEMISTRY; METALLURGY
C07K2317/10
CHEMISTRY; METALLURGY
G01N2800/367
PHYSICS
International classification
C07K16/26
CHEMISTRY; METALLURGY
Abstract
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.
Claims
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.
Description
(1) The invention will be understood more clearly by means of the following examples which are given by way of nonlimiting illustration, and also by means of
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EXAMPLES
Example 1: Cloning of DNA Fragments Corresponding to the Whole Sequence of Human AMH or to Truncated Sequences, and Transient Transfection in HEK 293T Cells
(6) 1.1. Gene Constructs
(7) The whole AMH sequence expressed is that of human AMH which comprises 560 amino acids (SEQ ID No. 2 corresponding to accession No. P03971 of the Uniprot KB database); this construct is called AMH-560. It comprises the signal peptide (amino acids 1-18) and a precursor portion (amino acids 19-25) which are cleaved during the post-translational maturation of the protein.
(8) Three other protein constructs, called AMH-1, AMH-2 and AMH-3, corresponding respectively to the sequences of amino acids 1-156 (SEQ ID No. 11), 1-255 (SEQ ID No. 8) and 1-451 (SEQ ID No. 5) of AMH were prepared. A polyhistidine (8-His) tag was added on the C-terminal side for these 4 constructs in order to facilitate purification (Tag not added in the sequence listing).
(9) The DNA fragments corresponding to the AMH-1, AMH-2, AMH-3 and whole AMH constructs, the sequences of which 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, AMH-3 and whole 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 obtained were verified by sequencing at the level of the inserts in order to be sure that they did not contain errors.
(10) TABLE-US-00005 TABLE 5 AMH Corresponding DNA fragment Name Polynucleotide SEQ ID No. X-8HIS-stop codon AMH-1 SEQ ID No. 86 SEQ ID NO. 86-CACCACCATCATCACCATCACCAC-TGA AMH-2 SEQ ID No. 87 SEQ ID NO. 87-CACCACCATCATCACCATCACCAC-TGA AMH-3 SEQ ID No. 88 SEQ ID NO. 88-CACCACCATCATCACCATCACCAC-TGA Whole AMH SEQ ID No. 89 SEQ ID NO. 89-CACCACCATCATCACCATCACCAC-TGA
(11) 1.2. Transient Transfection in HEK293T Cells
(12) Culture. The HEK-293T/17 SF cells (ATCC ACS-4500™) were cultured serum-free in HEK Plus SFM medium (ATCC #8006386597) enriched with glutamine (Gibco #250030.24), according to the supplier's instructions. The cells were cultured in F75 culture flasks and maintained in an incubator at 37° C. with 5% CO.sub.2 before transfection.
(13) Transfection. The HEK293T SF cells (10.sup.6 cells) were transfected by nucleofection using the Amaxa nucleofector device (Lonza), by applying the protocol supplied with the Amaxa cell line nucleofector kit V (#VCA-1003) and using 5 μg of DNA for 1 million cells per transfection. Briefly, 1 million cells are centrifuged at 200 g for 10 minutes in order to harvest them. The cell pellet is then resuspended in 100 μl of solution V (supplied in the kit). 5 μg of plasmid is added to the cell suspension. The whole mixture is gently mixed and transferred into an Amaxa cuvette (also supplied in the kit). The cuvette is inserted into the nucleofector device set to the Amaxa Q-001 program and the nucleofection is then activated. The sample is then immediately transferred to a warm medium in a 6-well cell culture plate that will be incubated at 37° C., 5% CO.sub.2. The HEK293T SF cells are cultured for 48 hours.
(14) Lysis and harvest. 48 Hours post-transfection, the supernatants are collected and then frozen at −80° C. after protease inhibitors (cOmplete™, EDTA-free protease inhibitor cocktail tablets from Roche) have been added. The transfected cell pellets (6×10.sup.6 cells/pellet) are taken up with 1.8 ml of 5.5 mM phosphate lysis buffer containing 130 mM NaCl, 0.5% Triton X-100, 5 U/ml benzonase nuclease (Novagen), 0.48 G/L MgCl.sub.2 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 is then placed in ice for 30 minutes, then centrifuged for 15 minutes at 13 000 g, 4° C. The supernatants obtained contain the AMH-1, AMH-2, AMH-3 and whole AMH proteins, and are stored at −80° C.
(15) 1.3. Western-Blot Analysis of the Protein Expression
(16) A first characterization of the expression products obtained during step 1.2. was carried out by SDS-PAGE analysis on a NuPAGE® Bis-Tris 4-12% gel in NuPAGE® MES SDS buffer (Life Technologies). Before being loaded onto the gel (14 μl/well), the transfection supernatants and lysates were diluted in the NuPAGE® LDS 4× Sample Buffer 4× (Life Technologies) (3/1, volume/volume) and underwent various treatments. The reduction is carried out by addition of a final concentration 55 mM of dithiothreitol (DTT). The heating is for 5 min at 75° C.
(17) Condition A: HEATED and REDUCED (with DTT)
(18) Condition B: HEATED and NON-REDUCED (without DTT).
(19) After the migration of the gel, the proteins separated by electrophoresis are transferred onto a 0.45 μm nitrocellulose membrane at 350 mA constant current for 50 minutes in a 1× Tris-glycine buffer containing 20% of methanol. The passivation of the membrane is carried out in the presence of 3% BSA (bovine serum albumin) in 5.5 mM phosphate buffer containing 130 mM NaCl, overnight at + 2/8° C. After passivation, a mouse anti-histidine monoclonal antibody (Qiagen, Cat. No. 34660) is diluted to 1/2000 in 5.5 mM phosphate buffer containing 130 mM NaCl and 0.05% of Tween 20, then 10 ml of this dilution are incubated with the membrane for 1 h 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 anti-β-actin monoclonal antibody (clone AC-15, Life Technologies, Cat. No. AM4302) in place of the anti-histidine antibody. This membrane serves to verify that, in each well, comparable amounts of total protein have been subjected to analysis (equivalent mode control).
(20) After rinsing of the membranes in order to remove the unbound antibodies (5 washes for 5 min in 5.5 mM phosphate buffer containing 130 mM NaCl and 0.05% Tween 20), they are incubated for 1 h with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Jacskon Immunoresearch, Cat. No. 115-036-003) diluted to 1/20000 in 1× PBS containing 0.2% Tween 20. After 5 washes of 5 minutes in 1× PBS containing 0.2% Tween 20, the visualization is carried out by incubating the membranes in a Clarity Western-blot Substrate solution (Biorad, Cat. No. 170-5061), for 5 minutes with stirring, before acquisition by chemiluminescence (Chemidoc XRS, Biorad).
(21) The results are presented in
(22) In conclusion, this Western blot analysis shows that the AMH-1, AMH-2, AMH-3 and whole AMH plasmid constructs clearly make it possible to express proteins containing a histidine tag, the apparent molecular weights of which correspond to the expected molecular weights, by virtue of the nucleotide sequences.
Example 2: Stable Expression of the AMH Protein in CHO Cells and Purification
(23) A stable clone of recombinant CHO (Chinese Hamster Ovary) cells expressing the whole AMH was obtained using the cGPS CHO-Sa CEMAX kit from Cellectis (Cat. No. CHOSa-0011-05 and CHOSa-0011-10) and according to the associated protocol. This kit allows the targeted intrachromosomal integration of an exogenous gene in CHO cells. It is composed of the cGPS CHO-Sa cell line (adherent cells), an integration vector into which the gene of interest is cloned, a vector constitutively expressing meganuclease I-Sce I and the TransMessenger™ Transfection Reagent kit (Qiagen Cat. No. 301525). The genetically modified cGPS CHO-Sa cell line has the particularity of containing, in its genome, a large and unique particular site recognized by meganuclease I-Sce I which has an endonuclease activity.
(24) 2.1. Cloning
(25) A synthetic gene encoding the whole human AMH protein (aa 1-560) linked to a C-terminal 8-histidine tag was ordered from the company Geneart. The gene was optimized for expression in the CHO host. This AMH gene was cloned into the pIM.LP2.Zeo integration vector sold by Cellectis, between the Eco RI and Not I sites under the control of the CMV promoter. The pIM.LP2.Zeo integration vector has the particularity of containing 2 regions which are homologous, respectively, to the region upstream and downstream of the unique site recognized by meganuclease I-Sce I in the CHO genome. These 2 particular regions frame the multiple cloning site into which the gene encoding the AMH was inserted. Furthermore, this integration plasmid provides the transfected CHO cells with 2 selective advantages. The first is the zeocin-resistance gene, controlled by the CMV promoter. The second is the neomycin resistance gene controlled by the SV40 promoter. This gene also enables resistance to geneticin (G418), an antibiotic close to neomycin.
(26) 2.2. Transfection
(27) One day before transfection (D-1), 2×10.sup.5 cGPS CHO-Sa CEMAX adherent cells are seeded, per 10 cm Petri dish, in an F-12K medium supplemented with 2 mM L-glutamine, penicillin (100 UI/ml), streptomycin (100 μg/ml), amphotericin B (Fongizone) (0.25 μg/ml) and 10% of fetal calf serum.
(28) On the day of transfection (D), 1 μg of the integration vector (pIM.LP2.Zeo) containing the AMH gene and 1 μg of meganuclease mRNA are diluted in EC-R buffer (available in the TransMessenger™ Transfection reagent kit). 4 μl of the Enhancer reagent are then added [nucleic acid (mg)/Enhancer (ml) ratio=½]. The total reaction volume must be 100 μl. The solution is then incubated for 5 minutes at ambient temperature. 16 μl of the TransMessenger™ reagent are subsequently added and then the whole mixture is incubated for 10 minutes at + 18/25° C., before being deposited onto the 2×10.sup.5 cells, the culture medium of which has been replaced beforehand with 900 μl of serum-free and antibiotic-free F-12k medium.
(29) 2.3 Clonal Selection and Characterization of Clones
(30) The AMH recombinant clones were selected according to their resistances to zeocin and to geneticin (G418).
(31) 24 h after transfection (D+1), the culture medium is replaced with 10 ml of complete medium supplemented with 0.6 mg/ml of G418, then, starting from the 6.sup.th day (D+6), the culture medium is regularly replaced with fresh medium supplemented with 0.6 mg/ml of G418 and 0.4 mg/ml of zeocin. 15 days after transfection (D+15), the cells are isolated by limiting dilution in a 96-well plate. After this cell cloning, the clones were amplified by culture and then tested: by PCR on the genomic DNA extracted from the CHO cells, using primers specific for the 5′ and 3′ ends of the AMH gene, by Western blotting in order to verify the expression of the AMH protein.
(32) Among the clones that were positive both in PCR and in Western blotting, the AMH5 3F1 clone was selected. For this clone, the PCR product obtained by amplification of the AMH gene was sequenced in its entirety in order to verify the integrity of the gene and to be sure that no mutation had been introduced during the selection.
(33) 2.4. Expression of the Recombinant AMH Using the Stable CHO-AMH5 3F1 Clone The AMH5 3F1 clone is cultured in a 225 cm.sup.2 culture dish in a proportion of 9×10.sup.6 cells in 60 ml of Excell 302 culture medium (Sigma Cat. No. 4324C) supplemented with 1% v/v antibiotics and antifungals (Gibco Cat. No. 15240), 12 mM L-glutamine (Gibco Cat. No. 25030), 6 g/l glucose (Sigma Cat. No. G8769), 0.4 mM iron citrate (Sigma Cat. No. F6129), 2% hypoxanthine-thymidine (Gibco Cat. No. 41065), 1% glycerol and 1 mg/l pepstatin A (Sigma Cat. No. P4265). The culture dishes are incubated in an incubator at 37° C. under an atmosphere of 7.5% CO.sub.2. After 4 days of culture (amplification), the cell suspension obtained (approximately 66×10.sup.6 cells) is re-seeded into 7 F225 culture dishes according to the same protocol. The supernatants of these cultures are harvested, then centrifuged at 5000 g and frozen at −25° C. until purification.
(34) 2.5. Purification of the Recombinant AMH Protein CHO-AMH5 3F1
(35) The culture supernatants previously harvested are thawed and pooled. A 5-liter volume of supernatant is then filtered on a 0.8 μm membrane (Nalgene, VWR Cat. No. 7345084) and then a 0.22 μm membrane (Nalgene aPES Rapid Flow). The sample is then concentrated 20-fold on hollow fiber having a cut-off threshold of 30 kDa (GE Cat. No. 564110-18). The retentate of approximately 100 ml is then diafiltered against 5 times its volume with a 50 mM potassium sodium phosphate buffer, pH7.8, containing 100 mM NaCl, 1 mM EDTA, 0.9 g/l azide. Two Complete EDTA free protease inhibitor tablets (Roche Cat. No. 11 873 580 001) are added. This retentate is purified by affinity chromatography on a Hi-trap NHS Sepharose column coupled to an anti-AMH (polyclonal or monoclonal) antibody in a proportion of 10 mg of antibody per ml of gel according to the supplier's protocol (GE Cat. No. 17-0716-01). The affinity column is equilibrated in diafiltration buffer, then 50 ml of retentate are injected onto the column at a speed of 0.5 ml/min. Washing is then carried out in 50 mM Tris HCl buffer pH 7.9, containing 100 mM NaCl, 1 mM EDTA. The elution of the AMH protein is then carried out with a 0.1 M glycine-HCl buffer, pH 2.9, at a speed of 0.5 ml/min. The elution fractions collected are immediately neutralized to pH 7-8, protease inhibitors are added thereto, and the fractions are pooled and stored at −80° C. in a 0.2 M NaHCO.sub.3 storage buffer containing 0.5 M NaCl, pH 7.5, and 20% ethanol.
Example 3: Preparation of Anti-AMH Antibodies of the Invention by Immunization of Mice
(36) 3.1. Immunogens
(37) The whole AMH and AMH-3 plasmids obtained in example 1 were amplified by culture in E. coli bacteria, then purified using the EndoFree Plasmid Mega Kit from Qiagen (Cat. No. 12381) or equivalent kit. For the preparation of the Gene Gun cartridges (Bio-Rad), 2 μg of plasmid DNA were precipitated on 0.22 mg of gold beads 1 μm in diameter in the presence of CaCl.sub.2 and of spermidine, according to the supplier's instructions. The beads thus prepared can be stored at + 2/8° C. in the dark, in the presence of a moisture absorber (desiccant sachet).
(38) The recombinant AMH protein CHO-AMH5 3F1 obtained in example 2 was mixed volume for volume with Freund's adjuvant (Sigma), prepared in the form of a water-in-oil emulsion and which is known to have a good adjuvant capacity. This preparation was carried out extemporaneously before each injection.
(39) 3.2. Immunizations
(40) The immunization experiments were carried out in female BALB/c (H-2.sup.d) mice from six to eight weeks old at the time of the first immunization. Various protocols are carried out: 4 doses of 4 μg per injection of whole AMH DNA at 0, 2, 4 and 6 weeks, 4 doses of 4 μg per injection of AMH-2 DNA at 0, 2, 4 and 6 weeks, 4 doses of 4 μg per injection of AMH-3 DNA at 0, 2, 4 and 6 weeks, 3 doses of 10 μg per injection of AMH protein at 0, 2, and 4 weeks.
(41) For the DNA immunizations, the mice were shaved on their abdomen. The Helios Gene Gun Delivery System (Bio-Rad) was used at a pressure of 2750 kPa to inject the DNA-coated gold beads into the skin of the mice. For the protein immunizations, the injection was carried out subcutaneously.
(42) In order to monitor the appearance of the antibodies, blood samples are regularly taken from the mice. The presence of the anti-AMH antibodies in these sera is tested by carrying out an ELISA on a 96-well microplate. The recombinant AMH protein CHO-AMH5 3F1 is used in capture mode (1 μg/well); after saturation, this antigen is reacted with various dilutions of sera to be tested (incubation at 37° C., for 1 h). The anti-AMH antibodies present in the serum are revealed using an AffiniPure goat anti-mouse IgG antibody conjugated to alkaline phosphate (H+L, Jacskon Immunoresearch, Cat No. 115-055-146), which binds to the antibodies sought (0.1 μg/well). The mice which had developed anti-AMH antibodies are thus identified among the immunized mice.
(43) Between 50 and 70 days after the first injections, the mice that had developed an anti-AMH humoral response were restimulated by means of an intravenous injection of 100 μg of AMH protein.
(44) 3.3. Preparation of the Hybridomas
(45) Three days after this final injection, the responding mice were sacrificed: the blood and spline were removed. The splenocytes obtained from the spline were cultured with Sp2/0-Ag14 myeloma cells in order for them to fuse and to become immortalized, according to the protocol described by Kohler and Milstein (Kohler and Milstein 1975, Kohler et al., 1976). After a culture period of 12-14 days, the hybridoma supernatents obtained were screened to determine the presence of anti-AMH antibodies using the ELISA assay described in the preceding section.
(46) 3.4. Selection of the Supernatants of Hybridomas Recognizing the 157-255 Region or the 256-451 Region of the Human AMH Protein
(47) A rabbit anti-6 histidine antibody (Sigma Aldrich, Cat. No. SAB4301134 or equivalent) was diluted in 1× PBS and adsorbed onto a 96-well microtitration plate in a proportion of 1 μg/well, by incubation overnight at + 18/25° C. The plate is then washed 3 times in PBS-0.05% Tween 20 buffer (PBS-T), then passivated by incubation in a PBS-T buffer containing 10 g/l of BSA and again washed 3 times in PBS-T buffer.
(48) The lysates obtained in example 1 and stored at −80° C. are thawed and diluted between ½ and 1/10 in 1× PBS buffer. 100 μl of each of the lysates (negative control, AMH-1, AMH-2, AMH-3 and whole AMH) are dispensed into several wells of the plate and incubated for 2 h at 37° C. The plate is then emptied and washed 4 times in PBS-T buffer containing 300 mM of NaCl. The hybridoma supernatants to be tested (100 μl) are then added to each of the 5 different lysates and incubated for 1 h at 37° C.
(49) After a further step of 4 washes in PBS-T containing 300 mM NaCl, the secondary antibody, which is an AffiniPure anti-mouse IgG produced in goats, conjugated to peroxidase (H+L, Jacskon Immunoresearch, Cat No. 115-035-166), is added. After a further step of 3 washes in PBS-T containing 300 mM NaCl, the reaction is visualized by incubating the plate for 10 min at + 18/25° C. in the presence of the SureBlue™ TMB Microwell Peroxidase substrate (KLP, Cat. No. 52-00-01). The plate is read by measuring the OD at 450 and 630 nm.
(50) Selection of the supernatants of hybridomas recognizing the 157-255 region. The hybridomas selected from the results of the ELISA are those of which the supernatants contain antibodies which recognize the AMH-2 lysate and do not recognize the AMH-1 lysate. Of course, the AMH-3 and whole AMH lysates are also recognized by the supernatants of the hybridomas chosen. The 5G5 1B11 hybridomas were thus selected.
(51) Selection of the supernatants of hybridomas recognizing the 256-451 region. The hybridomas selected from the results of the ELISA are those of which the supernatants contain antibodies which recognize the AMH-3 lysate and do not recognize the AMH-2 lysate, or the AMH-1 lysate. Of course, the whole AMH lysate is also recognized by the supernatants of the hybridomas chosen. The 3H8, 4C7 and 4G10 hybridomas were thus selected.
(52) After the selection, the hybridomas chosen were cloned according to the limiting dilution technique, well known to those skilled in the art, in order to be sure of the clonality. It was thus possible to obtain monoclonal hybridomas secreting the following anti-AMH antibodies: 5G5A10 and 1B11B1 which recognize the 157-255 region of human AMH, 3H8E2, 4C7E12 and 4G10E12 which recognize the 256-451 region of human AMH.
(53) The large-scale production of the monoclonal antibodies was carried out by culturing the hybridomas in the miniPERM™ bioreactor, according to a protocol derived from the publication by Falkenberg (1998). The monoclonal antibodies were then purified from the culture supernatant by affinity chromatography on protein A.
Example 4: Selection of the Anti-AMH Monoclonal Antibodies Recognizing the Non-Linear Epitopes
(54) Among the monoclonal antibodies obtained in example 3, those which were directed against linear epitopes were determined in order to select those recognizing non-linear epitopes. To do this, 80 synthetic peptides covering the whole of the amino acid sequence of human AMH were synthesized. The binding of the antibodies to each of these peptides were tested by ELISA.
(55) 4.1. Peptide Synthesis
(56) 80 Peptides of 16 amino acids were synthesized. Among the 16 amino acids, 12 correspond to the AMH sequence and overlap by 5 amino acids, thus covering the whole of the human AMH sequence (amino acids 1-560). A biotin and then an SGSG sequence (spacer arm) were added at the N-terminal end in order to facilitate the analysis of these peptides by ELISA. These peptides are non-purified and in solution (water/acetonitrile). They were verified by LC/MS mass spectrometry. The sequences synthesized are presented in table 6.
(57) TABLE-US-00006 TABLE 6 Sequences of the human AMH peptides Peptide AMH peptide No. (SEQ ID No.) Positions Total sequence 1 MRDLPLTSLALV 1-12 Bio-SGSG-MRDLPLTSLALV-amide (SEQ ID No. 90) 2 SLALVLSALGAL 8-19 Bio-SGSG-SLALVLSALGAL-amide (SEQ ID No. 91) 3 ALGALLGTEALR 15-26 Bio-SGSG-ALGALLGTEALR-amide (SEQ ID No. 92) 4 TEALRAEEPAVG 22-33 Bio-SGSG-TEALRAEEPAVG-amide (SEQ ID No. 93) 5 EPAVGTSGLIFR 29-40 Bio-SGSG-EPAVGTSGLIFR-amide (SEQ ID No. 94) 6 GLIFREDLDWPP 36-47 Bio-SGSG-GLIFREDLDWPP-amide (SEQ ID No. 95) 7 LDWPPGSPQEPL 43-54 Bio-SGSG-LDWPPGSPQEPL-amide (SEQ ID No. 96) 8 PQEPLCLVALGG 50-61 Bio-SGSG-PQEPLCLVALGG-amide (SEQ ID No. 97) 9 VALGGDSNGSSS 57-68 Bio-SGSG-VALGGDSNGSSS-amide (SEQ ID No. 98) 10 NGSSSPLRVVGA 64-75 Bio-SGSG-NGSSSPLRVVGA-amide (SEQ ID No. 99) 11 RVVGALSAYEQA 71-82 Bio-SGSG-RVVGALSAYEQA-amide (SEQ ID No. 100) 12 AYEQAFLGAVQR 78-89 Bio-SGSG-AYEQAFLGAVQR-amide (SEQ ID No. 101) 13 GAVQRARWGPRD 85-96 Bio-SGSG-GAVQRARWGPRD-amide (SEQ ID No. 102) 14 WGPRDLATFGVC 92-103 Bio-SGSG-WGPRDLATFGVC-amide (SEQ ID No. 103) 15 TFGVCNTGDRQA 99-110 Bio-SGSG-TFGVCNTGDRQA-amide (SEQ ID No. 104) 16 GDRQAALPSLRR 106-117 Bio-SGSG-GDRQAALPSLRR-amide (SEQ ID No. 105) 17 PSLRRLGAWLRD 113-124 Bio-SGSG-PSLRRLGAWLRD-amide (SEQ ID No. 106) 18 AWLRDPGGQRLV 120-131 Bio-SGSG-AWLRDPGGQRLV-amide (SEQ ID No. 107) 19 GQRLVVLHLEEV 127-138 Bio-SGSG-GQRLVVLHLEEV-amide (SEQ ID No. 108) 20 HLEEVTWEPTPS 134-145 Bio-SGSG-HLEEVTWEPTPS-amide (SEQ ID No. 109) 21 EPTPSLRFQEPP 141-152 Bio-SGSG-EPTPSLRFQEPP-amide (SEQ ID No. 110) 22 FQEPPPGGAGPP 148-159 Bio-SGSG-FQEPPPGGAGPP-amide (SEQ ID No. 111) 23 GAGPPELALLVL 155-166 Bio-SGSG-GAGPPELALLVL-amide (SEQ ID No. 112) 24 ALLVLYPGPGPE 162-173 Bio-SGSG-ALLVLYPGPGPE-amide (SEQ ID No. 113) 25 GPGPEVTVTRAG 169-180 Bio-SGSG-GPGPEVTVTRAG-amide (SEQ ID No. 114) 26 VTRAGLPGAQSL 176-187 Bio-SGSG-VTRAGLPGAQSL-amide (SEQ ID No. 115) 27 GAQSLCPSRDTR 183-194 Bio-SGSG-GAQSLCPSRDTR-amide (SEQ ID No. 116) 28 SRDTRYLVLAVD 190-201 Bio-SGSG-SRDTRYLVLAVD-amide (SEQ ID No. 117) 29 VLAVDRPAGAWR 197-208 Bio-SGSG-VLAVDRPAGAWR-amide (SEQ ID No. 45) 30 AGAWRGSGLALT 204-215 Bio-SGSG-AGAWRGSGLALT-amide (SEQ ID No. 118) 31 GLALTLQPRGED 211-222 Bio-SGSG-GLALTLQPRGED-amide (SEQ ID No. 119) 32 PRGEDSRLSTAR 218-229 Bio-SGSG-PRGEDSRLSTAR-amide (SEQ ID No. 46) 33 LSTARLQALLFG 225-236 Bio-SGSG-LSTARLQALLFG-amide (SEQ ID No. 120) 34 ALLFGDDHRCFT 232-243 Bio-SGSG-ALLFGDDHRCFT-amide (SEQ ID No. 121) 35 HRCFTRMTPALL 239-250 Bio-SGSG-HRCFTRMTPALL-amide (SEQ ID No. 47) 36 TPALLLLPRSEP 246-257 Bio-SGSG-TPALLLLPRSEP-amide (SEQ ID No. 122) 37 PRSEPAPLPAHG 253-264 Bio-SGSG-PRSEPAPLPAHG-amide (SEQ ID No. 123) 38 LPAHGQLDTVPF 260-271 Bio-SGSG-LPAHGQLDTVPF-amide (SEQ ID No. 61) 39 DTVPFPPPRPSA 267-278 Bio-SGSG-DTVPFPPPRPSA-amide (SEQ ID No. 124) 40 PRPSAELEESPP 274-285 Bio-SGSG-PRPSAELEESPP-amide (SEQ ID No. 125) 41 EESPPSADPFLE 281-292 Bio-SGSG-EESPPSADPFLE-amide (SEQ ID No. 126) 42 DPFLETLTRLVR 288-299 Bio-SGSG-DPFLETLTRLVR-amide (SEQ ID No. 127) 43 TRLVRALRVPPA 295-306 Bio-SGSG-TRLVRALRVPPA-amide (SEQ ID No. 128) 44 RVPPARASAPRL 302-313 Bio-SGSG-RVPPARASAPRL-amide (SEQ ID No. 129) 45 SAPRLALDPDAL 309-320 Bio-SGSG-SAPRLALDPDAL-amide (SEQ ID No. 130) 46 DPDALAGFPQGL 316-327 Bio-SGSG-DPDALAGFPQGL-amide (SEQ ID No. 131) 47 FPQGLVNLSDPA 323-334 Bio-SGSG-FPQGLVNLSDPA-amide (SEQ ID No. 132) 48 LSDPAALERLLD 330-341 Bio-SGSG-LSDPAALERLLD-amide (SEQ ID No. 62) 49 ERLLDGEEPLLL 337-348 Bio-SGSG-ERLLDGEEPLLL-amide (SEQ ID No. 63) 50 EPLLLLLRPTAA 344-355 Bio-SGSG-EPLLLLLRPTAA-amide (SEQ ID No. 64) 51 RPTAATTGDPAP 351-362 Bio-SGSG-RPTAATTGDPAP-amide (SEQ ID No. 133) 52 GDPAPLHDPTSA 358-369 Bio-SGSG-GDPAPLHDPTSA-amide (SEQ ID No. 65) 53 DPTSAPWATALA 365-376 Bio-SGSG-DPTSAPWATALA-amide (SEQ ID No. 66) 54 ATALARRVAAEL 372-383 Bio-SGSG-ATALARRVAAEL-amide (SEQ ID No. 134) 55 VAAELQAAAAEL 379-390 Bio-SGSG-VAAELQAAAAEL-amide (SEQ ID No. 135) 56 AAAELRSLPGLP 386-397 Bio-SGSG-AAAELRSLPGLP-amide (SEQ ID No. 136) 57 LPGLPPATAPLL 393-404 Bio-SGSG-LPGLPPATAPLL-amide (SEQ ID No. 137) 58 TAPLLARLLALC 400-411 Bio-SGSG-TAPLLARLLALC-amide (SEQ ID No. 138) 59 LLALCPGGPGGL 407-418 Bio-SGSG-LLALCPGGPGGL-amide (SEQ ID No. 139) 60 GPGGLGDPLRAL 414-425 Bio-SGSG-GPGGLGDPLRAL-amide (SEQ ID No. 140) 61 PLRALLLLKALQ 421-432 Bio-SGSG-PLRALLLLKALQ-amide (SEQ ID No. 141) 62 LKALQGLRVEWR 428-439 Bio-SGSG-LKALQGLRVEWR-amide (SEQ ID No. 67) 63 RVEWRGRDPRGP 435-446 Bio-SGSG-RVEWRGRDPRGP-amide (SEQ ID No. 68) 64 DPRGPGRAQRSA 442-453 Bio-SGSG-DPRGPGRAQRSA-amide (SEQ ID No. 142) 65 AQRSAGATAADG 449-460 Bio-SGSG-AQRSAGATAADG-amide (SEQ ID No. 143) 66 TAADGPCALREL 456-467 Bio-SGSG-TAADGPCALREL-amide (SEQ ID No. 144) 67 ALRELSVDLRAE 463-474 Bio-SGSG-ALRELSVDLRAE-amide (SEQ ID No. 145) 68 DLRAERSVLIPE 470-481 Bio-SGSG-DLRAERSVLIPE-amide (SEQ ID No. 146) 69 VLIPETYQANNC 477-488 Bio-SGSG-VLIPETYQANNC-amide (SEQ ID No. 147) 70 QANNCQGVCGWP 484-495 Bio-SGSG-QANNCQGVCGWP-amide (SEQ ID No. 148) 71 VCGWPQSDRNPR 491-502 Bio-SGSG-VCGWPQSDRNPR-amide (SEQ ID No. 149) 72 DRNPRYGNHVVL 498-509 Bio-SGSG-DRNPRYGNHVVL-amide (SEQ ID No. 150) 73 NHVVLLLKMQVR 505-516 Bio-SGSG-NHVVLLLKMQVR-amide (SEQ ID No. 151) 74 KMQVRGAALARP 512-523 Bio-SGSG-KMQVRGAALARP-amide (SEQ ID No. 152) 75 ALARPPCCVPTA 519-530 Bio-SGSG-ALARPPCCVPTA-amide (SEQ ID No. 153) 76 CVPTAYAGKLLI 526-537 Bio-SGSG-CVPTAYAGKLLI-amide (SEQ ID No. 154) 77 GKLLISLSEERI 533-544 Bio-SGSG-GKLLISLSEERI-amide (SEQ ID No. 155) 78 SEERISAHHVPN 540-551 Bio-SGSG-SEERISAHHVPN-amide (SEQ ID No. 156) 79 HHVPNMVATECG 547-558 Bio-SGSG-HHVPNMVATECG-amide (SEQ ID No. 157) 80 VPNMVATECGCR 549-560 Bio-SGSG-VPNMVATECGCR-amide (SEQ ID No. 158)
(58) 4.2. ELISA
(59) The 96-well microplates are coated with streptavidin (10 μg/ml, 1 μg/well) in a 1× PBS buffer for 1 h at 37° C., and then passivated in a PBS-0.05% Tween 20 buffer (PBS-T) containing 10 g/l of BSA overnight at ambient temperature. The passivation solution is removed, then the biotinylated peptides are dispensed (10 μg/ml in 1× PBS buffer, 1 μg per well) and incubated for 1 h at 37° C. After 4 washes in PBS-T, the monoclonal antibody to be tested is added at a concentration of 1 μg/ml. After incubation for 1 h 30 at 37° C. and 4 PBS-T washes, a peroxidase-conjugated anti-mouse
(60) IgG antibody is added. The visualization is carried out with the TMB substrate with measurement of the optical density (OD) at 450 nm.
(61) In order to validate the ELISA format, a commercial anti-AMH antibody was used as positive control. It is the AA011 clone from the company AnshLabs. This antibody specifically recognizes peptide 52 (SEQ ID No. 137), the OD signal obtained is greater than 1. Among the 5 monoclonal antibodies obtained in example 3, the 4C7E12 clone also recognizes peptide 52. The 4C7E12 clone therefore recognizes the same epitope as the AA011 clone. It is not retained. The monoclonal antibodies 5G5A10, 1B11B1, 3H8E2 and 4G10E12 recognize none of the 80 AMH peptides tested. Their epitopes are not therefore linear. It is these four non-linear antibodies 5G5A10, 1B11B1, 3H8E2 and 4G10E12 which were selected for developing a method for quantifying the AMH protein in the biological samples.
Example 5: Characterization of the Anti-AMH Antibodies 5G5A10, 1B11B1, 3118E2 and 4G10E12
(62) 5.1. Comparison of the Capture Capacity of the Anti-AMH Antibodies 5G5A10, 1B11B1, 3H8E2 and 4G10E12 by ELISA
(63) The capture antibodies (5G5A10, 1B11B1, 3H8E2 and 4G10E12) diluted to 5 μg/ml in 200 mM Tris, pH 6.2, are dispensed into a 96-well microtitration plate in a proportion of 0.5 μg/well and incubated for 1 h 30 at 37° C. The plate is then washed 3 times in a PBS-0.05% Tween 20 buffer (PBS-T), then passivated for 2 h by incubation in a PBS-T buffer containing 10 g/l of BSA and again washed 3 times in PBS-T buffer.
(64) The lysates obtained in example 1 and stored at −80° C. are thawed, and diluted to 1/20 in 1× PBS. 100 μl of each of the lysates (negative control, AMH-1, AMH-2, AMH-3 and whole AMH) are dispensed and incubated overnight at 37° C. The plate is then emptied and washed 4 times in PBS-T buffer containing 300 mM of NaCl. A biotinylated anti-histidine antibody (Qiagen Cat. No. 34440) diluted to 1/1000 in passivation buffer is dispensed and then incubated for 1 h 30 at 37° C. The plate is again washed 3 times, before incubation with the streptavidin-peroxidase conjugate (Jackson Immunoresearch Cat. No. 016-030-034) diluted to 1/2000 in passivation buffer, for 1 h 30 at 37° C. After a further step of 3 washes in PBS-T, the reaction is visualized by incubating the plate for 10 min at + 18/25° C. in the presence of the SureBlue™ TMB Microwell Peroxidase substrate (KLP, Cat. No. 52-00-01). The plate is read by measuring the OD at 450 and 630 nm.
(65) The results obtained are presented in table 7. The OD values measured for the negative controls that are PBS and lysate transfected without plasmid are at most 0.28, which corresponds to the nonspecific signal.
(66) TABLE-US-00007 TABLE 7 Capture capacity of the anti-AMH antibodies (OD.sub.450 nm value) Ab 1B11B1 Ab 5G5A10 Ab 3H8E2 Ab 4G10E12 PBS neg 0.129 0.141 0.196 0.246 0.137 0.132 0.178 0.122 control Lysate neg 0.199 0.189 0.21 0.223 0.165 0.159 0.168 0.281 control AMH-1 lysate 0.221 0.236 0.257 0.266 0.244 0.198 0.218 0.203 AMH-2 lysate 4.244 4.246 4.316 4.191 0.176 0.197 0.184 0.168 AMH-3 lysate 3.683 3.774 3.186 3.365 1.859 1.815 1.801 1.622 Whole AMH 3.184 3.351 3.296 3.36 1.667 1.711 2.132 1.812 lysate
(67) The results in table 7 show that the 1B11B1 and 5G5A10 antibodies capture the AMH-2, AMH-3 and whole AMH lysates (OD >3). The 3H8E2 and 4G10E12 antibodies capture the AMH-3 and whole AMH lysates (OD >1.6). The AMH-1 lysate (amino acids 1-156) is not captured by any of the monoclonal antibodies tested.
(68) Furthermore, the 1B11B1 and 5G5A10 antibodies which recognize the 157-255 region of the human AMH protein have capture capacities greater than those of the 3H8E2 and 4G10E12 antibodies which recognize the 256-451 region. Indeed, in the case of recognition (underlined values in table 2), for a given lysate, the OD values obtained with the 1B11B1 and 5G5A10 antibodies are higher than those obtained by the 3H8E2 and 4G10E12 antibodies. It will therefore be judicious to preferentially use, in capture mode, the 1B11B1 antibody or the 5G5A10 antibody.
(69) 5.2. Western-Blot Characterization
(70) In order to better characterize the reactivities of the anti-AMH monoclonal antibodies, Western-blot analyses were carried out.
(71) Protein purification. Transfections were carried out according to the protocol of example 1 with the plasmids encoding the AMH-2 and AMH-3 constructs, and also a negative control (without plasmid). The culture supernatants are recovered and the AMH proteins which are found therein are purified by batchwise metal-chelate affinity chromatography, by virtue of their polyhistidine tags. To do this, the transfection supernatants are incubated overnight at + 2/8° C. with stirring with an Ni-NTA resin (Roche, Cat. No. 115-26-70) equilibrated in 11 mM phosphate buffer containing 260 mM NaCl, pH 7.4. The resin is then recovered by centrifugation for 3 min at 3000 g and the supernatant containing the non-absorbed material is removed. After 3 washes with the equilibration buffer, the protein is eluted with a 5.5 mM phosphate buffer containing 130 mM NaCl, pH 7.4, and containing 500 mM of imidazole, and protease inhibitors, and the pH of which is adjusted to 7.6. Elution is carried out by incubation at + 2/8° C. with stirring, overnight. The supernatant containing the AMH is then harvested after centrifugation of the resin for 3 minutes at 3000 g. This purification step is necessary in order to concentrate the AMH proteins and especially to remove the large amounts of BSA present in the supernatants, which disrupt the migration of the SDS-PAGE gels.
(72) Western blot. This analysis was carried out according to the protocol described in example 1, section 1.3, either using the anti-histidine antibody, or replacing it with an anti-AMH antibody (1 μg per membrane).
(73) Results. The results of this analysis are presented in
Example 6: Detection of AMH by Sandwich Immunoassay
(74) On commercial AMH kits, a significant difference was observed between the concentrations obtained for a sample as a function of the time at which the assay was carried out after the sample had been taken. This type of variability is unacceptable in clinical practice, it is therefore essential to have available a robust kit, for which the AMH concentration measured is accurate and reproducible and which allows the samples to be stored at + 2/8° C. or at −19/−31° C. before assaying. Such an assay was carried out by choosing an antibody directed against amino acids 157-255 of human AMH.
(75) 6.1. Automated Immunoassay Procedure
(76) The detection of the AMH in the biological samples was carried out by one-step sandwich immunoassay using the VIDAS® automated immunoanalysis device (bioMérieux). The single-use tip acts both as solid phase for the reaction and its pipetting system. The cartridge of the automated device is composed of 10 wells (X0 to X9) covered with a sealed and labeled aluminum foil. The first well (X0) comprises a portion that is precut in order to facilitate the introduction of the sample. The last well (X9) is an optical cuvette in which the fluorescence of the substrate is measured. The various reagents required for the analysis are contained in the intermediate wells. All the steps of the assay are thus carried out automatically by the instrument. They consist of a succession of cycles of suction/discharge of the reaction medium.
(77) Sensitization and passivation of the tips. The tips were sensitized with 270 μl of a solution of 5G5A10 monoclonal antibody for the pair C1 or of 8C5B10H5 monoclonal antibody for the pair C2, each at 7 μg/ml in a Tris buffer, pH 6.2. After approximately 20 h of incubation at + 18/25° C. with the sensitizing solution, the tips were emptied. 300 μl of this same solution containing 5 g/l of bovine albumin are then added for the passivation of the tips at + 18/25° C. for approximately 20 h. The tips are then emptied, dried, then stored at +4° C. until use, away from moisture.
(78) Preparation of the conjugated antibody solutions. For the pair C1, the conjugate solution contains the 4G10E12 monoclonal antibody, in the form of a Fab′ fragment, coupled to alkaline phosphatase. For the pair C2, the conjugate solution contains the 5G5A10 monoclonal antibody, in the form of a Fab′ fragment, coupled to alkaline phosphatase. The conjugated antibodies were diluted to approximately 0.1 μg/ml in a Tris/NaCl BSA buffer, pH 6.5.
(79) Immunoassay. As soon as the VIDAS® tip is in contact with the sample, the immunological reaction begins since the capture antibodies are immobilized on this tip. The automated device mixes the sample to be tested (89.6 μl) with 226 μl of the conjugate solution. The incubation lasts approximately 10 minutes at 37° C. and enables the specific binding of the AMH to, on the one hand, the antibody adsorbed onto the cone and to, on the other hand, the conjugated antibody (detection antibody). The unbound components are then removed by means of 3 washes with a 54 mM Tris buffer, pH 7.3, containing 154 mM NaCl and 0.55% Tween 20. During the final step of visualization, the 4-methylumbelliferyl phosphate substrate is suctioned and then discharged in the tip; the enzyme of the conjugated antibodies catalyzes the reaction for hydrolysis of this substrate to 4-methylumbelliferone, the emitted fluorescence of which is measured at 450 nm. The value of the fluorescence signal (RFV=relative fluorescence value) is proportional to the concentration of the antigen present in the sample.
(80) 6.2. AMH Assaying of the Samples Having Experienced Various Storage Conditions
(81) Eight pools of natural samples from women between the ages of 19 and 52 were assayed in parallel with the commercial AMH Gen II Assay kit (Beckman Coulter) and with two pairs of antibodies (C1 and C2) on the VIDAS® automated device. Each sample pool consists of 3 sera from women that come from an Etablissement Français du Sang (EFS) [French Blood Bank] of the Rhône Alps region.
(82) These samples were tested less than 4 hours after the taking of the samples corresponding to the time T0, then after 24 hours of storage at + 2/8° C., after 7 days of storage at + 2/8° C. and after 7 days of storage at −19/−31° C. corresponding to the various storage conditions of these samples.
(83) For the kit in microplate form, the respective doses were calculated as a function of the range recommended by the producer. The ratios of the doses obtained for each storage condition relative to the dose obtained at T0 were then calculated. For the VIDAS® automated device, the fluorescence signals (RFV=Relative fluorescence value) obtained with each of the 2 pairs of antibodies, for each storage condition, were used to calculate the ratios with respect to the fluorescence signals obtained at T0. The results obtained are presented in table 8.
(84) TABLE-US-00008 TABLE 8 ratio of the AMH doses or signals obtained after storage under various conditions and at T0 (T/T0 ratio). 7 days at 24 h at +2/8° C. 7 days at +2/8° C. −19/−31° C. C1 C2 REF C1 C2 REF C1 C2 REF Pool 001 0.99 1.00 1.10 0.95 1.06 1.14 0.98 0.99 1.20 Pool 002 0.98 1.04 1.27 0.96 1.13 1.20 0.99 1.03 1.30 Pool 003 0.98 1.00 1.14 0.96 1.04 1.05 1.01 1.02 1.07 Pool 004 1.01 0.99 1.58 0.97 1.08 1.57 0.99 1.02 1.73 Pool 005 0.96 1.02 1.46 0.95 1.09 1.33 0.97 1.01 1.24 Pool 006 1.00 1.01 1.35 0.97 1.10 1.40 0.97 0.99 1.35 Pool 007 0.97 1.03 1.43 0.96 1.11 1.66 0.95 1.01 1.51 Pool 008 0.99 1.06 1.34 0.95 1.07 1.34 1.00 1.03 1.35 Mean 0.99 1.02 1.33 0.96 1.09 1.34 0.98 1.01 1.34 Min 0.96 0.99 1.10 0.95 1.04 1.05 0.95 0.99 1.07 Max 1.01 1.06 1.58 0.97 1.13 1.66 1.01 1.03 1.73 C1: VIDAS ® assay with 5G5A10 as capture antibody and 4G10E12 as detection antibody; C2: VIDAS ® assay with 8C5B10H5 as capture antibody and 5G5A10 as detection antibody; REF: AMH Gen II microplate assay.
(85) The closer to the T/T0 ratio is to 1, the less variability there is, over time or according to the storage conditions, in the amounts of AMH detected. The AMH assays using the C1 or C2 pair always give the same result, regardless of the prior storage conditions of the sample: the T/T0 ratio is 0.99 (C1) and 1.02 (C2) on average when the sample was stored for 24 h at + 2/8° C. These values are very close to 1, whereas the reference kit displays a mean ratio of 1.33 indicating that the dose measured is on average multiplied by 1.33. The same type of observation is made for the other storage conditions tested.
(86) In summary, the 5G5A10 antibody recognizes a non-linear epitope of the 157-255 zone of human AMH, the 4G10E12 antibody recognizes a non-linear epitope of the 256-451 zone of human AMH, the 8C5B10H5 antibody recognizes a linear epitope (aa 508-519 of human AMH) in the mature region of AMH, the antibodies of the AMH Gen II Assay kit are both directed against linear epitopes of the mature region.
(87) The antibody pairs C1 and C2 make it possible to develop AMH immunoassays which are robust and the result of which does not vary as a function of the prior storage conditions of the sample.
Example 7: Analytical Sensitivity of VIDAS® AMH
(88) The analytical sensitivity of the VIDAS® AMH immunoassay (pair C1) described in example 6 was compared to that of assays according to the prior art. In order to be able to compare the antibody pairs under similar conditions, the entire experiment was carried out on the VIDAS® automated device. The tips were coated either with the 5G5A10 antibody (antibody recognizing the C-terminal portion of the pro region of AMH), or with the AA012 antibody (commercial antibody—AnshLabs) according to the procedure explained in example 6. For the detection step, two conjugates were compared: the 4G10E12 antibody and the AA011 antibody (commercial antibody—AnshLabs). Both were conjugated to alkaline phosphatase according to the procedure of example 6. The 4G10E12 conjugate was used at 180 ng/ml, the AA011 conjugate at 500 ng/ml. The 4G10E12 conjugate, which recognizes the C-terminal portion of the pro region of AMH, is thus used in a much lower amount compared with the commercial antibody, which is entirely unexpected.
(89) A standard range ranging from 0.055 to 11 ng/ml of AMH was prepared by diluting sera for which the AMH concentrations were known, in serum from a menopausal woman (negligible AMH concentration), then measured using the following 3 assay formats:
(90) Pair C1: capture 5G5A10+detection 4G10E12
(91) AnshLabs pair: capture AA012+detection AA011
(92) Mixed pair: capture 5G5A10+detection AA011
(93) The results are presented in table 9.
(94) TABLE-US-00009 TABLE 9 Comparison of the RFV signals and of the S/N ratios obtained with various antibody pairs by VIDAS ® for the standard range VIDAS RFV Signal VIDAS RFV/RFV0 Capture 5G5A10 AA012 5G5A10 5G5A10 AA012 5G5A10 Detection 4G10E12 AA011 AA011 4G10E12 AA011 AA011 (conc ng/ml) (180) (500) (500) (180) (500) (500) AMH ng/ml RFV RFV RFV S/N S/N S/N 0 1 45 7 0.055 51 81 53 51 2 8 0.11 92 113 91 92 3 13 0.275 224 209 221 224 5 32 0.9 916 735 910 916 16 130 2.1 2113 1602 2097 2113 36 300 4 3817 2938 3816 3817 65 545 6.3 4839 3915 4891 4839 87 699 8.7 5672 4810 5764 5672 107 823 11 6335 5392 6404 6335 120 915 S/N = “signal/noise” ratio. For a given assay format, ratio of the RFV signal to the RFV signal of point 0 of the range (without AMH).
(95) The pair C1 (5G5A10 & 4G10E12) exhibits excellent signal dynamics and an extremely low background noise (1 RFV at point 0). The pair according to the prior art AA012 & AA011 exhibits poorer signal dynamics (5392 RFV for 11 ng/ml, instead of 6335 RFV for the pair C1) and especially a considerable background noise of 45 RFV. The mixed pair (5G5A10 & AA011) has signal dynamics comparable to the pair C1 (same capture antibody). The background noise (7 RFV) is considerably lower than for the pair AA012 & AA011, but remains higher than for the pair C1. As can be seen, in terms of the signal/noise (S/N) ratios, the assay that is analytically the most sensitive is the one which uses the pair C1, then the one with the mixed pair, and in last place the one with the AnshLabs pair.
Example 8: Analytical Specificity of the VIDAS AMH Assay
(96) The analytical specificity of the VIDAS AMH assay (pair C1) described in example 6 was established by analyzing compounds with cross reactivity. These compounds were added in overload amounts to serum samples containing 1 ng/ml and 4 ng/ml of AMH. The results of this study are summarized in table 10:
(97) TABLE-US-00010 TABLE 10 Cross reactivities of the VIDAS ® AMH assay Compound tested Concentration tested % Cross reactivity Activin A 100 ng/ml 0.10% Inhibin A 100 ng/ml 0.12% LH 500 IU/l 0.21% FSH 500 IU/l 0.23%
(98) No significant cross reactivity was detected at the concentrations tested. The VIDAS AMH assay exhibits excellent analytical specificity.
Example 9: Accuracy of the VIDAS AMH Assay
(99) The study of accuracy of the VIDAS AMH assay (pair C1) was carried out using a panel of human samples representative of 5 concentration levels of the measurement range. For each concentration level, the repeatability (intra-series accuracy), the intra-batch accuracy and the intra-laboratory accuracy (intra-instrument inter-batch accuracy) were estimated. The values obtained during this study are reported in table 11:
(100) TABLE-US-00011 TABLE 11 Accuracy of the VIDAS AMH assay N (number Concentration % CV of level % CV % CV Intra- repetitions) (ng/ml) Repeatability Intra-batch laboratory 519 0.22 4.1 6.6 8.3 520 1.08 4.4 8.0 9.9 520 2.99 4.4 7.4 9.8 520 5.45 4.8 7.6 8.9 520 7.37 4.4 8.2 10.6
(101) As this very wide study shows, the VIDAS AMH assay exhibits good reproducibility: the coefficient of variation (CV) between different batches does not exceed 11%.
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