WO2014204327A1 - Assay for anti-mullerian hormone - Google Patents
Assay for anti-mullerian hormone Download PDFInfo
- Publication number
- WO2014204327A1 WO2014204327A1 PCT/NZ2014/000123 NZ2014000123W WO2014204327A1 WO 2014204327 A1 WO2014204327 A1 WO 2014204327A1 NZ 2014000123 W NZ2014000123 W NZ 2014000123W WO 2014204327 A1 WO2014204327 A1 WO 2014204327A1
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- WO
- WIPO (PCT)
- Prior art keywords
- amh
- proamh
- binding agent
- assay
- antibody
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Definitions
- the present invention provides assays, methods and kits for measuring, for example, an amount of an Anti-Mullerian Hormone (AMH) in a sample.
- AMH Anti-Mullerian Hormone
- the present invention provides assays, methods and kits for measuring different forms of AMH, including, for example, proAMH and AMH N(C , in biological samples obtained from human and non-human animals.
- AMH Anti-Mullerian Hormone
- MIS Mullerian Inhibiting Substance
- AMH contributes to the virilisation of the brain and behaviour of mice, via a hormonal mechanism (Morgan et al. (2011) Behav Brain Res 221 : 304-306; Wang et al. (2009) Proc Natl Acad Sci USA 106 :7203-7208; Wittmann & McLennan (2011) J Neuroendocrinol 23 :831-838) .
- a boy's level of AMH inversely correlates with a measure of his matu ration (Morgan et al. (2011) PLoS One 6:e20533) and with the severity of his symptoms, if he has an autism spectrum disorder (Pankhurst & McLennan (2012) Transl Psychiatry 2 :el48).
- AMH is the first ovarian secretion to cease during the perimenopausal transition (van Rooij et al. (2005) Fertil Ster/7 83 :979-987) .
- AMH is a hormone in adults, then there are obvious im plications for the u nderstanding of menopause and the existence of sex biases in age-related conditions.
- AMH is synthesised as a glycosylated (Budzik et al. (1980) Cell 21 :909-915; Lorenzo et al. (2002) J Chromatogr B Biomed Sci Appl 766 :89-98; Picard et al. (1986) FEBS Letters 195 : 73-76; Picard et al. (1978) Mol Cell Endocrinol 12 : 17-30) cysteine-linked homodimer of the 560 amino acid preproprotein (preAMH) (Cate et al. (1986) Cell 45 :685-698). The first 24 amino acids are removed during synthesis yielding the prohormone (proAMH) (Cate et al.
- AMH C is the receptor-activating component of total AMH (MacLaughlin et al. (1992) Endocrinology 131 : 291-296; di Clemente et al. (2010) Mol Endocrinol 24 :2193-2206) .
- Some recombinant forms of AMH also contain alternatively cleaved AMH, which yields an extended C-terminal peptide consisting of amino acids 255-560 (AM H255-560) (Ragin et al. (1992) Protein Expr Purif 3 : 236-245) .
- the AMH in blood is generally presumed to be the bioactive cleaved form based on an historic investigation of a sex-cord tumour patient with pathologically elevated levels of AMH (Ragin et al. (1992) Protein Expr Purif 3 :236-245).
- the present invention provides assays, methods and kits capable of detecting and measuring different forms of AMH in a biological sample, and in particular, proAMH and proAMH N , c .
- the present invention provides assays, methods and kits for the detection and measurement of the different species of AMH present in a biological sample, and in particular a biological sample such as a whole blood sample, plasma and serum.
- an assay for proAMH in a biological sample comprising proAMH and AMH N , C obtained from a subject, the assay comprising measuring the level of proAMH in the sample using a first binding agent, a second binding agent and a splitting reagent, wherein :
- splitting reagent splits the AMH N ⁇ C complex into AMH N and AMH C ;
- the second binding agent when the first binding agent binds to the C-terminal domain of proAMH and AMH N(C , the second binding agent is specific for and binds to the N-terminal domain of proAMH .
- the proAMH assay according to this aspect of the present invention further comprises:
- the proAMH assay according to this aspect of the present invention further comprises:
- an assay for AMH N;C in a biological sample comprising proAMH and AMHN,C obtained from a subject comprising measuring the level of AMH N)C in the sample using a first binding agent, a second binding agent and a splitting reagent, wherein the first binding agent binds to proAMH and ⁇ , the second binding agent binds to AMH N or AMH C but does not bind to proAMH or AMH N/C , and wherein the splitting reagent is sufficient to split the AMH N , C complex into AMH N and AMH C .
- the AMH N ,c assay further comprises:
- the level of bound AMH N or AMH C corresponds to the amount of AMH N(C in the sample.
- the AMH N(C assay further comprises:
- the level of bound AMH N or AMH C corresponds to the amount of AMH N ,c in the sample.
- binding agents and splitting reagent used in the assays described herein may also be used in methods for measuring the level of AMH in a biological sample, and in particular measuring the levels of proAMH and AMH N , c in a sample.
- a method for measuring the level of proAMH in a su bject comprising:
- the splitting reagent splits the AMH N ,c complex into AMH N and AMH C , and wherein when the first binding agent binds to the N-terminal domain of proAMH and AMH N , C , the second binding agent is specific for and binds to the C-terminal domain of proAMH, or wherein when the first binding agent binds to the C-terminal domain of proAMH and AMH N
- the method further comprises:
- the method for measu ring proAMH according to this aspect of the present invention further comprises:
- a method for measuring the level of AMH N;C in a subject comprising:
- the first binding agent binds to proAMH and AMH N , C
- the second binding agent binds to AMH N or AMHc but does not bind to proAMH or AMH N , C
- the splitting reagent is sufficient to split the AMH N
- the method further comprises:
- the level of bound AMH N or AMH C corresponds to the amount of AMH N ,c in the sample.
- the first binding agent is immobilised on a solid support.
- the second binding agent comprises a detectable label.
- the first binding agent is an antibody or an antigen binding fragment thereof and/or the second binding agent is an antibody or an antigen binding fragment thereof.
- the antibody or antigen binding fragment may be selected from the group consisting of a polyclonal, monoclonal, bispecific, chimeric and humanized antibody or an antigen binding fragment thereof, and in certain embodiments the antibody is a monoclonal antibody or antigen binding fragment thereof.
- the second binding agent comprises a labelled form of AMH N or AMH C , depending on the configuration of the assay or method (i.e. specificity of the first binding agent for the N- or C-terminal domain of AMH).
- the second binding agent is an antibody comprising a detectable label which binds to AMH C or AMH N .
- a detectable label for example, Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446) that specifically binds to AMH C .
- the splitting reagent is selected from the group consisting of a salt, a detergent, and a chemical reagent sufficient to alter pH.
- the splitting reagent comprises deoxycholate.
- the proAMH and AMH NiC assays and methods according to the present invention are configured in a format selected from the group consisting of an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a fluoroimmunoassay, an immunofluorometric assay, and an immunoradiometric assay.
- ELISA enzyme linked immunosorbent assay
- RIA radioimmunoassay
- fluoroimmunoassay a fluoroimmunoassay
- an immunofluorometric assay an immunoradiometric assay.
- the invention further provides assays and methods for measuring proAMH comprising binding members that are specific for proAMH.
- an assay for proAMH in a biological sample comprising proAMH and AMH N;C obtained from a subject, the assay comprising measuring the level of proAMH in the sample using a binding agent specific for proAMH.
- the proAMH assay according to this aspect of the present invention comprises:
- the present invention also provides a method for measuring proAMH in a subject, the method comprising measu ring the level of proAMH in a biological sample obtained from the subject using a binding member specific for proAMH .
- the binding agent specific for proAMH binds to an epitope that spans the cleavage site between the N- and C-terminal domains of proAMH .
- the present invention provides an assay for AMH N , C in a biological sample comprising proAMH and AMH NjC obtained from a subject, the assay comprising :
- the amount of AMH N , C in the sample represents the difference between total AMH and proAMH measured in the sample.
- the present invention also provides a method for measuring the amount of AMH N;C in a subject, the method comprising :
- a method for measuring the level of proAMH in a su bject comprising:
- the amount of AMH in the sample is determined using a conventional assay for AMH.
- the amount of proAMH in the sample is determined using a proAMH assay described herein.
- the present invention provides an assay for measu ring total AMH in a biological sample comprising proAMH and AMH N/C , the assay comprising :
- the total amount of AMH in the sample represents the sum of the amounts of proAMH and AMH N , C measured in the sample.
- the present invention also provides a method for measuring total AMH in a biological sample comprising proAMH and AMH N , C , the method comprising :
- the total amount of AMH in the sample represents the sum of the amounts of proAMH and AMH N ,c measured in the sample.
- the amou nt of proAMH in the sample is determined using an assay or method described herein.
- the amount of AMH N , c in the sample is determined using an assay or method described herein.
- a dual purpose assay for measu ring different species of AMH in a biological sample comprising any combination of the assays described herein .
- the dual purpose assay measures proAMH and AMH NiC .
- the present invention also provides a method for measuring proAMH and AMH N ,c in a subject, the method comprising any combination of the methods for measuring proAMH and AMH N , C described herein.
- kits comprising binding agents, splitting reagents and i nstructions for performing the assays and methods described herein.
- a kit for measu ring proAMH in a biological sample obtained from a su bject comprisi ng proAMH and AMH N ,c the kit comprising a first binding agent, a second binding agent, a splitting reagent and instructions for how to measu re the level of proAMH in the sample,
- the com ponents of the kit a re fu rther characterised :
- the splitting reag ent is capable of splitting the AMH N(C com plex into AMH N and AMH C ;
- AMH NiC in a biological sample comprising proAMH and AMH N , C obtai ned from a subject, the kit com prising a first binding agent, a second binding agent, a splitting reagent, and instructions for how to measure the level of AMH N)C in the sample.
- the components of the kit are fu rther characterised :
- splitting reagent splits the AMH N , C com plex into AMH N and AMH C ;
- the second bi nding agent is specific for and binds to AM H N or AMH C .
- the first binding agent is immobilised on a solid su bstrate.
- the second binding agent comprises a detectable label.
- the first bindi ng agent is an antibody or an antigen binding fragment thereof and/or the second bind ing agent is an antibody or an antigen bi nding fragment thereof.
- the antibody or antigen binding fragment may be selected from the g rou p consisti ng of a polyclonal, monoclonal, bispecific, chimeric and humanized antibody or an antigen binding fragment thereof, and in certai n embodiments the anti body is a monoclonal anti body or antigen binding fragment thereof.
- the second binding agent comprises a labelled form of AMH N or AMH C , depending on the configu ration of the assay (i .e. specificity of the first binding agent for the N- or C- terminal domain of AMH).
- the second binding agent is an antibody comprising a detectable label which binds to AMH C or AMH N , for example, Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446) which specifically binds to AMH C .
- the splitting reagent is selected from the group consisting of a salt, a detergent, and a chemical reagent sufficient to alter pH.
- the splitting reagent comprises deoxycholate.
- kits for measuring proAMH in a biological sample obtained from a subject comprising proAMH and AMH N , C , the kit comprising a binding agent for proAMH together with instructions for how to measure the level of proAMH in the sample.
- the binding agent is capable of binding to an epitope that spans the cleavage site between the N- and C-terminal domains of proAMH.
- the binding agent may be an antibody or antigen binding fragment thereof, and in particular a monoclonal antibody or antigen binding fragment thereof. Further, the binding agent may comprise a detectable label.
- Figure 1 shows a schematic illustration of the various AMH species, including preAMH, proAMH, AMH N , C , AMH N , AMH C , AMH2 55 -5 60 and AMH25-254 where numbers represent the position in the amino acid sequence with 1 being the initiating codon methionine. The ability of each species to bind to the canonical AMH receptors is indicated.
- FIG. 2 shows a schematic illustration of a conventional/commercial ELISA (top panel) and the proAMH ELISA (middle panel) and AMH N , C ELISA (lower panel) according to the present invention.
- the illustrated cleavage step involves separation of AMH N ,c into AMH and AMH C .
- the detection antibody in the lower panel does not bind to proAMH, most commonly because the epitope is masked by the N-terminal domain of proAMH.
- FIG 3 shows detection of proAMH, AMH N and AMH C .
- Unprocessed or furin-treated recombinant human AMH (rhAMH) was analyzed on western blots under reducing conditions, using antibodies against the N-terminal (panel A) or C-terminal (panel B) domain of AMH.
- the location of the proAMH, AMH N , AMH C and alternatively cleaved AMH (AMH255-560) are marked with arrows.
- the left lane in each blot shows the molecular weight markers.
- Figure 4 shows the non-specific bands associated with immunoprecipitation. Control goat IgG was added to human blood, and then precipitated.
- the precipitated proteins were analysed by western blots under reducing conditions, using only a secondary antibody.
- the expected location of the proAMH, AMH N , AMH C and alternatively cleaved AMH 2 55-56o are marked with arrows (see Fig. 3).
- the left lane shows the molecular weight markers.
- FIG. 5 shows proAMH and AMH N , C is present in human blood.
- AMH was immunoprecipitated with N-terminal (N-ip) or C-terminal (C-ip) specific antibodies from plasma samples obtained from a boy (Bl) with high AMH levels and a post-menopausal woman (PMW) to act as an AMH-deficient control (Table 1). Samples were run on western blot under reducing conditions, and detected with either anti-N terminal (A) or anti-C terminal antibodies (B).
- the proAMH, AMH N and ANH C bands are indicated by arrows, with the heavy chain and light chain IgG bands indicated by the upper and lower arrowheads, respectively.
- the molecular weight of AMH255-560 is indicated by the double arrowhead.
- the left lane in each blot shows the molecular weight markers, with the right lane a 5 ng rhAMHc control.
- Figure 6 shows an anti-C terminal antibody selectively immunoprecipitates AMH C .
- rhAMH proAMH, with lesser levels of AMH N , C , Fig. 3
- rhAMH c was immunoprecipitated with beads coated with either an anti-C terminal or anti-N terminal antibody of AMH. The precipitates were then analyzed by western blot under reducing conditions, using an anti-C antibody to detect AMH C and proAMH .
- anti-C terminal antibody precipitated AMHc (lane 2) but not proAMH (lane 3), whereas the anti-N terminal antibody precipitated proAMH (lane 4).
- Lanes 5 and 6 show that there is minimal precipitation of recombinant forms of AMH when the beads are not coated with AMH-specific antibodies.
- the strong bands indicated by arrowheads are the antibodies used for precipitation.
- the left lane (lane 1) shows the molecular weight markers.
- FIG 7 shows proAMH and AMH NjC is present in the blood of men, women and boys.
- AMH was immunoprecipitated with an anti-N-terminal antibody and the western blot was probed with both the C-terminal and N-terminal antibodies.
- Plasma samples were obtained from boys (B2-3), adult men (Ml-3), pre-menopausal women (Wl-3) and one post-menopausal woman (PMW).
- the proAMH, AMH N and AMH C bands are indicated by arrows, with the heavy chain and light chain IgG bands indicated by the upper and lower arrowheads, respectively.
- the molecular weight of AMH255-560 is indicated by the double arrowhead.
- the high-molecular weight bands are evident in low gain scans (A), with higher gain scan required to detect AMH C (B) (see also Fig. 8).
- the left hand lane shows the molecular weight markers.
- Figure 8 shows AMH N
- AMH C precipitated from the blood of adults is near the detection limit of a western.
- the samples from Fig. 7 were therefore run a second time to confirm the presence of AMH C .
- the orginal scan is illustrated in (A), and was quantified by densitometry (C).
- the original image was then enhanced linearly to give visual confirmation of the densitometry measurements (B).
- Figure 9 shows deglycosylation of AMH was conducted on AMH immunoprecipitated from a boy with a combination of 5 deglycosylating enzymes (PNGase F, Sialidase A, 0- glycosidase, 3(l ⁇ 4)-galactosidase and ⁇ - ⁇ -acetylglcuosaminidase).
- PNGase F Sialidase A
- 0- glycosidase 0- glycosidase
- 3(l ⁇ 4)-galactosidase ⁇ - ⁇ -acetylglcuosaminidase
- the immunoprecipitating antibody was covalently linked to the agarose beads to minimise any overlap between the IgG heavy chain and deglycosylated AMH N .
- the thick arrows indicate the position of the bands for proAMH and AMH N and the arrowhead indentifies the immunoprecipitating IgG heavy chain before deglycosylation treatment.
- Thin arrows indicate the new location of the proAMH and AMH N bands after treatment with deglycosylating enzymes.
- the left lane in each blot shows the molecular weight markers.
- FIG 10 shows AMH is glycosylated in adult samples.
- Deglycosylation of AMH was conducted on AMH immunoprecipitated from two men (M4 & M5) and two pre-menopausal women (W5 & W5) .
- a combination of deglycosylating enzymes was used; PNGase F, Sialidase A, O-glycosidase, p(l ⁇ 4)-galactosidase (3(l ⁇ 4)-Gal-ase) and ⁇ - ⁇ - acetylglcuosaminidase ( ⁇ -GlcNAc-ase).
- PNGase F Sialidase A
- O-glycosidase O-glycosidase
- p(l ⁇ 4)-galactosidase 3(l ⁇ 4)-Gal-ase
- ⁇ - ⁇ - acetylglcuosaminidase ⁇ -GlcNAc-
- M5 showed the strongest signal with a visible proAMH band at 72 kDa and two AMH N bands at ⁇ 60 kDa and both bands shifted after deglycosylation.
- W4 and W5 do not show bands before deglycosylation but do have faint proAMH and AMH N bands after deglycosylation. This is consistent with the sample from M5 and the boys in Fig. 9, where the anti-N-terminal antibody appears to have a higher affinity for degylcosylated proAMH and AMH N .
- FIG 11 shows proAMH binds to the ELISA matrix of a commercial AMH assay (Beckman Coulter; Catalogue #A79765).
- rhAMH c (c), rhAMH N (n) or rhAMH (AMH) were loaded onto the matrix from an AMH ELISA and onto a matrix from an Inhibin B (InhB) ELISA (Beckman Coulter; Catalogue #A81303).
- the proteins captured by the ELISAs were then recovered and anlayzed by western blots using either an anti-N terminal antibody or an anti-C terminal antibody.
- rhAMH c , rhAMH N and rhAMH were also run directly on the western, to act as standards.
- the proAMH, AMH C and AMH N bands are indicated.
- a nonspecific band of variable intensity was recovered from the AMH ELISA wells, and is indicated by arrowheads.
- the left hand lane of blot
- Figure 12 shows proAMH from human blood binds to the ELISA matrix of a commercial AMH assay (Beckman Coulter; Catalogue #A79765). Blood from 3 boys (B4- B6) were mixed with the commercial assay buffer and added to either AMH or Inhibin B (InhB) wells. The captured proteins were recovered and analyzed by western blots, using antibodies to the C-terminal and N-terminal. The non-specific band that is variably recovered from the wells is indicated by an arrowhead (see also Fig. 11). The left hand lane of each blot shows the molecular weight markers.
- Figure 13 shows that deoxycholate can prevent AMH N from being captured. Varying concentrations of dexoycholate was added to samples of rhAMH or rhAMH that had been treated with furin to produce rhAMH N(C . After incu bation, the samples were added to ELISA wells from a commercial assay (Beckman Coulter; Catalogue #A79765), and the captured proteins analyzed by western blots. The left hand lane shows the molecular weight markers.
- FIG 14 shows that deoxycholate can release AMH N captured from blood.
- AMH was captured from the serum, and then washed with either phosphate buffer (PB) or 0.2% sodium deoxycholate.
- PB phosphate buffer
- the serum in all 6 wells was from the same adult.
- the captured proteins were recovered and analyzed by western blots. The two columns on the right hand side of the gel did not have serum added.
- FIG. 15 shows an illustration of the AMH used in the proAMH assay in Fig. 16.
- rhAMH was incu bated with furin and an aliquot of the original rhAMH and the fu rin-treated AMH analyzed by western blots, using the anti-N terminal antibody.
- the rhAMH is predominantly proAMH, whereas the furin-treated rhAMH is heavily enriched AMH N ,c / with minor levels of residual proAMH present.
- Figure 16 shows demonstration of a proAMH -specific ELISA. Varying concentrations of rhAMH (proAMH) and furin-treated rhAMH (AMH N ,c) were assayed using a commercial assay (Beckman Coulter, Catalogue #A79765), with the added deoxycholate step.
- Figure 17 shows capture of proAMH and AMH N ,c using another commercial ELISA assay matrix (AnSH Labs AMH ELISA; Catalogue #AL-105-i) .
- rhAMH c , rhAMH N , rhAMH or human serum were loaded onto wells from an Ansh Labs AnshLite Ultrasensitive AMH/MIS chemiluniescent immunoassay.
- the proteins captured by the wells were then recovered an aniayzed by western blots using either an anti-N terminal antibody or an anti-C terminal antibody.
- the proAMH, AMH C , AMH N and AMH 2 s5-56o bands are indicated.
- a non-specific band of variable intensity was recovered from the wells, and is indicated by arrowheads.
- the left hand lane of each blot shows the molecular weight markers.
- the serum sample of each blot was initially separated by one lane (deleted) from the recombinant proteins.
- Figure 18 shows that proAMH ELISA detects AMH from human samples and there is no difference in detection compared to the rhproAMH initially used as a standard .
- Figure 19 shows detection of various forms of rh-AMH at 14 nM by the duoset ELISA (R&D systems, catalogue #DY1737) under normal running conditions (control) or when samples have been pre-treated with deoxycholate.
- the ELISA detects AMH C but not proAMH and only a small signal is given for AMH N
- c- Pre-treatment of AMH N(C with deoxycholate increases the signal to a level equivalent to AMH C but only has a minor effect on the proAMH preparation which contains small quantities of AMH N , C .
- Figure 20 shows recovery of various forms of AMH at 14 nM from the duoset ELISA (R&D systems, catalogue #DY1737) under normal running conditions (control) or when samples have been pre-treated with deoxycholate.
- AMH N , C does not bind to the ELISA capture antibody as efficiently as AMH C under normal conditions.
- Deoxycholate pre- treatment causes the amount of AMH c -binding in rh-AMH N ,c samples relative to the amount of purified AMH C binding.
- Figure 21 shows demonstration of an ELISA that detects AMH C or AMH N(C but not proAMH.
- Rh-AMH C R&D systems, catalogue #1737-MS was assayed to demonstrate that deoxycholate does not interfere with normal function of the assay.
- Figure 22 shows a sequence alignment between human, mouse, rat and bovine AMH sequences.
- Figure 23 shows total AMH concentration plotted against %proAMH in boys (A), men aged 24-65 (B) and men older than 65 years of age (C).
- Figure 24 shows Total AMH and %proAMH values in females.
- A Total AMH changes vs. age;
- B %proAMH vs. age and
- Anti-Mullerian Hormone is synthesised as a glycosylated cysteine-linked homodimer of the 560 amino acid prepropeptide (preAMH).
- preAMH prepropeptide
- the first 24 amino acids are removed during synthesis yielding the prohormone (proAMH), which does not activate AMH receptors, which includes, for example, Type II AMH Receptor (AMHRII).
- proAMH prohormone
- AMH receptors which includes, for example, Type II AMH Receptor (AMHRII).
- AMH C Type II AMH Receptor
- AMH C is the receptor-activating component of AMH
- Some recombinant forms of AMH also contain alternatively cleaved AMH, which yields an extended C-terminal peptide consisting of amino acids 255-560 (AMH 25 5-56o) . It is unclear whether this form of AMH binds to or activates AMH receptors.
- AMH NjC and AMH N have both been described as proAMH, as they are a precursor forms of AMH C .
- AMH NiC and AMH C have both been referred to as mature AMH.
- Fig. 1 is particularly instructive.
- Fig. 1 is particularly instructive.
- preAMH is intended to mean the large precursor or prepropeptide of AMH which includes a short signal sequence (24 amino acids) followed by the propeptide that forms homodimers.
- proAMH is intended to mean the prohormone that corresponds to a 140 kDa dimer of identical disilfide-linked 70 kDa monomer subunits; each monomer contains an N- terminal domain and a C-terminal domain.
- N-terminal domain of AMH when used in the context of a human AMH sequence is intended to mean residues 1-451 of the human prepropeptide or residues 25-451 if the leader sequence has been removed
- C-terminal domain of AMH when used in the context of a human AMH sequence is intended to comprise residues 452-560 the human propeptide.
- AM HN is intended to mean a dimer of identical N-terminal domains.
- AHc is intended to mean a dimer of identical C-terminal domains.
- AMH N ,c is intended to mean a non-covalent complex consisting of a 120 kDa N- terminal dimer (AMH N ) and a 25 kDa C-terminal dimer.
- AMH 25 5-56o is intended to mean an alternatively cleaved form of AMH which consists in an extended C-terminal peptide comprising amino acids 255-560.
- antibody refers to an immunoglobulin molecule capable of specifically binding an antigen, such as, for example, AMH, and typically by binding an epitope or antigenic determinant of AMH, such as, for example, a C-terminal or N-terminal region of AMH, including AMH N and AMH C .
- an antigen such as, for example, AMH
- an epitope or antigenic determinant of AMH such as, for example, a C-terminal or N-terminal region of AMH, including AMH N and AMH C .
- the term “antibody” broadly includes full length antibodies and antigen binding fragments or regions thereof. Also included are monoclonal and polyclonal antibodies, multivalent and monovalent antibodies, multispecific antibodies (for example bi-specific antibodies), chimeric antibodies, human antibodies, humanized antibodies and antibodies that have been affinity matured.
- an antibody binds selectively or specifically to a region or domain, such as the N- or C-terminal region of AMH, if the antibody binds preferentially to a region or domain of AMH which has, e.g. has less than 25%, or less than 10%, or less than 1% or less than 0.1% cross-reactivity with non- AMH antigens/epitopes or other non-target AMH species, when appropriate.
- the antibody will have a binding affinity (dissociation constant (Kd) value), for the antigen or epitope of about 10 "6 , or 10 "7 M, 10 "8 M, or 10 _9 M, or 10 "10 , or 10 "11 or 10 "12 M. Binding affinity may be assessed using surface plasma resonance, for example, or Scatchard analysis.
- an "antigen binding fragment” or “antibody fragment” or “binding fragment” when used in reference to an antibody means a portion of the intact antibody that preferably retains most or all, or minimally at least one of, the normal binding functions of the intact antibody.
- antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments, linear antibodies, diabodies, single chain antibodies (ScFV), domain antibodies and multispecific antibodies.
- N-terminal antibody when used in the context of AMH, means an antibody (or antigen binding fragment, antibody fragment or binding fragment) raised against the N-terminal domain of AMH (defined by amino acid residues 1-451 of the human propeptide) which specifically binds to an epitope or antigenic determinant of the N- terminal region of AMH.
- N-terminal antibody and “anti-N terminal antibody” may be used interchangeably throughout this specification and are intended to have the same meaning.
- C-terminal antibody when used in the context of AMH, means an antibody (or antigen binding fragment, antibody fragment or binding fragment) raised against the C-terminal domain of AMH (defined by amino acid residues 452-560 of the human propeptide) which specifically binds to an epitope or antigenic determinant of the C-terminal region of AMH.
- C-terminal antibody and “anti-C terminal antibody” may be used interchangeably throughout this specification and are intended to have the same meaning.
- domain and “region” when used in the context of a domain or region of a protein, may be used interchangeably.
- antibodies raised against the "N-terminal domain” or “N-terminal region” of AMH could be taken to have the same intended meaning. However, this is not always the case.
- N-terminal antibody could be understood to mean an antibody (or antigen binding fragment, antibody fragment or binding fragment) which has been raised against a region, such as an epitope or antigenic determinant, of the N-terminal domain of AMH.
- epitope includes any antigenic (e.g., a protein) determinant capable of specific binding to an antibody and/or a T cell receptor. That is, a site on an antigen to which B and/or T cells respond.
- Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
- An epitope typically includes at least 3, 5 or 8-10 amino acids. The amino acids may be contiguous, or non-contiguous amino acids juxtaposed by tertiary folding. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
- antigenic variant refers to polypeptide sequences different from the specifically identified sequences, wherein one or more amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of 1, 2, 3, 4, 5, 6, 10, 15, 20, 40, 50 or more amino acids are specifically contemplated. Variants may be naturally-occurring allelic antigenic variants, or non-naturally occurring antigenic variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, antigenic variants of the polypeptides useful in the invention have biological activities including hormone function or antigenic-binding properties that are the same or similar to those of the parent polypeptides.
- an antigenic variant of human AMH may include the non-human sequences of AMH, such as those AMH sequences derived from mouse, rat, sheep, bovine, pig etc (see, for example, Fig. 19).
- polypeptide antigenic variants may be identified by physical methods known in the art, for example, by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et a/. , Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) by recombinant DNA techniques also described by Sambrook et al. or by identifying polypeptides from natural sources with the aid of such antibodies.
- an “isolated antibody” is an identified antibody that has been separated or recovered, or both, from a component of its natural environment. For example, separated from proteins including enzymes and hormones.
- the antibody is purified to at least 95%, or 96% or 97% or 98% or 99% by weight of antibody. Purity can be determined by the Lowry method, for example. Ordinarily the antibody will be prepared by at least one purification step.
- a "monoclonal antibody” means an antibody that is a highly specific antibody directed against (or which binds to) a single antigen target.
- a monoclonal antibody may be obtained from a population of homogenous or substantially homogenous antibodies wherein each monoclonal antibody is identical and/or bind the same epitope, except for natural mutations that may occur in minor amounts.
- Monoclonal antibodies are prepared using methods known the art, such as, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, and Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly and individually referred to herein as Harlow and Lane) .
- polyclonal antibody means an antibody which may be directed against (or which may bind to) multiple antigen targets.
- Polyclonal antibodies are prepared using methods known the art (such as, for example, in Harlow and Lane, ibid).
- binding agent refers to any solid or non-solid material capable of binding a species of AMH, fragment or an antigenic variant thereof. In one embodiment the term refers to any natural or non-natu ral molecule that binds to a species of AMH, fragment or antigenic variant thereof. Examples of binding agents include proteins, peptides, nucleic acids, carbohydrates, lipids, and small molecule compounds. One selective or specific binding agent is an antibody or antigen binding fragment thereof.
- sample or “biological sample” as used herein means any sample taken or derived from a subject. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from blood being assessed, for example, for ovarian reserve in a subject, and used to evaluate blood for the levels of Anti-Mullerian Hormone (AMH) . Included are samples taken or derived from any subjects such as from normal healthy subjects and/or healthy subjects for whom it is useful to understand the levels of AMH. Preferred samples are body fluid samples.
- AMH Anti-Mullerian Hormone
- body fluid sample refers to a sample of bodily fluid obtained for the purpose of, for example, diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient. In certain embodiments, such a sample may be obtained for the purpose of determining ovarian reserve.
- the sample may be any sample known in the art in which AMH can be detected.
- any body fluids such as a whole blood sample, plasma, serum, ovarian follicular fluid sample, seminal fluid sample, cerebrospinal fluid, saliva, sputum, urine, pleural effusions, interstitial fluid, synovial fluid, lymph, tears, for example, although whole blood sample, plasma, serum, ovarian follicu lar fluid sample and seminal fluid sample are particularly suited for use in this invention.
- body fluid samples would be more readily analysed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
- purified does not require absolute purity. Purified refers in one embodiment to at least 90%, or 95%, or 98%, or 99% homogeneity of, to provide an example, of a polypeptide or antibody in a sample.
- subject as used herein is preferably a mammal and includes human, and non-human mammals such as cats, dogs, horses, cows, sheep, deer, mice, rats, primates (including gorillas, rhesus monkeys and chimpanzees), possums and other domestic farm or zoo animals.
- non-human mammals such as cats, dogs, horses, cows, sheep, deer, mice, rats, primates (including gorillas, rhesus monkeys and chimpanzees), possums and other domestic farm or zoo animals.
- the assays, methods and kits described herein have application to both human and non-human animals, in particular, and without limitation, humans, primates, farm animals including cattle, sheep, goats, pigs, deer, alpacas, llamas, buffalo, companion and/or pure bred animals including cats, dogs and horses.
- Preferred subjects are humans, and most preferably "patients" who as used herein refer to living humans who may receive or are receiving medical care or assessment for a disease or condition. Further, while a subject is preferably a living organism, the invention described herein may be used in postmortem analysis as well.
- ELISA as used herein means an enzyme linked immunosorbent assay, a type of competitive binding assay comprising antibodies and a detectable label used to quantitate the amount of an analyte in a sample.
- capture antibody means an antibody which is typically immobilized on a solid support such as a plate, bead or tube, and which antibody binds to and captures analyte(s) of interest, for example proAMH or AMH N ,c or both.
- detection antibody means an antibody comprising a detectable label that binds to analyte(s) of interest.
- the label may be detected using routine detection means for a quantitative, semi-quantitative or qualitative measure of the analyte(s) of interest, for example proAMH or AMHN.C or both.
- the term "relating to the presence or amou nt" of an analyte reflects that assay signals are typically related to the presence or amou nt of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest.
- an assay is "configured to detect” an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte.
- an analyte is measured in a sample.
- a level “higher” or “lower” than a control, or a "change” or “deviation” from a control (level) in one embodiment is statistically significant.
- a higher level, lower level, deviation from, or change from a control level or mean or historical control level can be considered to exist if the level differs from the control level by about 5% or more, by about 10% or more, by about 20% or more, or by about 50% or more compared to the control level.
- Statistically significant may alternatively be calculated as P ⁇ 0.05.
- Higher levels, lower levels, deviation, and changes can also be determined by recourse to assay reference limits or reference intervals. These can be calculated from intuitive assessment or non-parametric methods. Overall, these methods may calculate the 0.025, and 0.975 fractiles as 0.025* (n+1) and 0.975 (n+1). Such methods are well known in the art. Presence of a marker absent in a control may be seen as a higher level, deviation or change. Absence of a marker present in a control may be seen as a lower level, deviation or change.
- AMH conventional assay for AMH
- BC ELISA Beckman Coulter AMH Generation II ELISA Kit
- ANSH Labs AMH ELISA ANSH ELISA; Catalogue #A79765
- AMH in blood namely the bioactive cleaved form, AMH N ,c, which does not vary with age or gonadal pathology.
- AMH N ,c the bioactive cleaved form
- the present invention therefore provides assays, methods and kits for the detection and measurement of the different species of AMH present in a biological sample, and in particular a biological sample such as a whole blood sample, plasma or serum.
- rhAMH recombinant human AMH
- the molecular weights and immunoreactivity of proAMH, AMH N , AMH C and AMH255-560 were investigated by western blot under reducing-conditions using antibodies against the N- and C-terminal regions of AMH (Fig. 3; Example 1).
- the recombinant human form of AMH (rhAMH) derived from HEK cells was predominantly a 72 kDa species, with lesser amounts of other forms of AMH observed.
- the apparent molecular weight for proAMH is larger than predicted from its amino acid sequence due to its glycosylation state (Figs. 9 and 10; Example 5).
- proAMH The 72 kDa species is designated proAMH because it bound both the C- and N- terminal antibodies, and because pretreatment with the proprotein convertase furin diminished the intensity of the band. Conversely, the intensity of the AMH C and AMH N bands increased following furin treatment, providing further confirmation of their identities, proAMH was more efficiently detected by the N-terminal than the C-terminal antibody (Fig. 3A versus Fig. 3B; Example 1). AMH C at 12.5 kDa was detected exclusively by the C- terminal antibody (Fig. 3B), whereas no equivalent band was detected using the N-terminal antibody (Fig. 3A)
- AMH is a glycosylated protein, and the two AMH N bands resolve to a single band when AMH is deglycosylated (data not shown).
- the apparent molecular weight of the AMH N band is larger than predicted from amino acid sequence, due to its glycosylation.
- AMH in blood is generally presumed to be AMH N(C , and over 400 scientific papers have been published on this basis. Applicants now provide the first direct evidence to show that blood contains a mixture of AMH species, and predominantly proAMH and AMH N(C .
- the level of AMH in blood is too low to be detected by direct western blot analysis, necessitating enrichment of AMH species, by techniques known in the art.
- enrichment of AMH species from a sample is performed by immunoprecipitation.
- Immunoprecipitation is a technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. Broadly, this process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point during the precipitation process, such as, to highly porous sepharose or agarose resins or slurries, or to non/magnetic beads, for example.
- An artefact of immunoprecipitation is the inclusion of non-specific bands which appear in western blots from the precipitating reagents. Accordingly, the use of this technique necessitates that the non-specific bands do not obscure the bands of the target protein(s) to be analysed.
- non-specific bands were identified by adding control IgG from a non-immunised goat to human serum.
- the resulting bands were different in size to proAMH, AMH N and AMH C (compare Figs. 3 and 4; Example 2).
- Faint non-specific bands were detected at approximately 47 kDa (Fig. 4). These bands would obscure trace but not significant levels of ⁇ 255-560 ⁇
- Applicants Having validated an approach to enrichment of AMH through immunoprecipitation, Applicants then sought to investigate the species of AMH present in blood. It is widely acknowledged, and known to a person skilled in the art, that boys possess higher blood levels of AMH compared to, for example, prepubescent girls, or adult men or women. Accordingly, the initial analysis of blood AMH was performed using plasma samples obtained from pre-pubescent boys. Since post-menopausal women (PMW) only possess trace levels of AMH (Table 1 ; Example 4) a plasma sample derived from a PMW subject was used as a negative control.
- PMW post-menopausal women
- AMH C suggests it is coimmunoprecipitating as part of the AMH N ,c complex because a detection antibody to the N-terminal domain does not directly bind to AMH C (Fig. 5A) .
- N-terminal antibody to AMH from plasma samples obtained from an additional two pre- pubescent boys (B2, B3), three young men (Ml, M2, M3) and three pre-menopausal women
- Example 4 As shown in Figure 7, proAMH and AMH N was detected in the blood of boys, men and women with the intensity of the bands being broadly commensurate with the estimate of proAMH levels as quantified by ELISA (Fig 7; Table 1) .
- the PMW control had no detectable levels of AMH N and only trace levels of proAMH (Fig. 7).
- Applicants have developed a novel approach to specifically measure proAMH in a biological sample comprising different forms of AMH, including proAMH and AMH N;C .
- a general concept underlying one aspect of the present invention i.e. proAMH specific assay
- Fig. 2 contrasted against conventional AMH ELISA assay platforms.
- this schematic shows that conventional ELISAs do not distinguish between proAMH and AMH N , C , in other words conventional ELISAs measure both proAMH and AMH N , C , whereas, in one aspect, the proAMH assay according to the present invention is specific for proAMH. This observation is supported by experimental evidence presented in Examples 6-8 and Figures 11-18 which follow.
- an assay for proAMH in a biological sample comprising proAMH and AMH N(C obtained from a subject, the assay comprising measuring the level of proAMH in the sample using a first binding agent, a second binding agent and a splitting reagent, wherein:
- splitting reagent splits the AMH N(C complex into AMH N and AMH C ;
- the proAMH assay further comprises:
- the proAMH assay uses the non-covalent association between the cleaved AMH species, namely the AMH N ,c complex, to distinguish between proAMH and AMH N(C .
- a first binding agent in this case antibody
- a solid support such as, for example, an ELISA plate well, and is used to capture proAMH and AMH N , C present in a biological sample such as plasma or serum.
- splitting reagent is then introduced to the assay to split the AMH N , C complex into AMH N and AMH C . Since, in this example, the capture antibody only binds to the N-terminal domain of AMH, splitting of the AMH N ,c non-covalent complex via a splitting reagent causes dissociation of AMH C , which is subsequently removed by further wash step(s).
- a second binding agent in this case an antibody
- This detection antibody is specific for proAMH, since the AMH NjC complex has been split and AMH C , which previously formed part of the AMH N , C complex, has been washed away and is no longer present.
- Antibodies for example, polyclonal, monoclonal, bispecific, chimeric and humanized antibodies and antigen binding fragments are particularly preferred examples of binding agents according to the present invention.
- the first binding agent is bound to and immobilized on a solid support.
- Solid supports include, for example, multi-well plates, beads and tubes, and the binding agents according to the present invention may be conjugated directly to the solid support, for example, or by indirect binding.
- antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface. This is described in more detail below under the heading "Immunoassay".
- the detection antibody may comprise a detectable label.
- the second binding agent comprises a detectable label.
- Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
- the presence of the label may be quantified using standard techniques known to a person skilled in the art.
- the amount of detection antibody is directly proportional to the amount of proAMH present in the sample, thereby providing a quantative measure of the amount of
- the capture antibody in relation to the specific assay configuration depicted in Fig. 2 could be specific for the N-terminal or the C-terminal domain of AMH, provided that the detection antibody binds to the opposite domain of AMH.
- the detection antibody binds to the opposite domain of AMH.
- the capture antibody binds to the N-terminal domain of AMH
- the detection antibody must bind to the C- terminal domain.
- the capture antibody binds to the C-terminal domain of AMH, then the detection antibody must bind to the N-terminal domain.
- the assay for proAMH comprises a first binding agent which binds to the C-terminal domain of AMH and a second binding agent which binds to N-terminal domain of AMH.
- the first binding agent binds to the N-terminal domain of AMH and the second binding agent binds to the C-terminal domain of AMH.
- the splitting reagent is used to split AMH N(C into AMH N and AMH C .
- the splitting reagent must be capable of splitting non-covalent complexes.
- Examples of splitting reagents according to the present invention include, but are not limited to, salts, detergents, and chemical reagents sufficient to alter pH, such as acids or bases. Specific examples of splitting reagents include sodium deoxycholate, acetic acid and sodium dodecyl sulphate.
- the splitting reagent will not split proAMH into its constituent N- and C- terminal domains since these domains are covalently linked through a polypeptide bond. Accordingly, the bound proAMH will bind the detection antibody when added to the assay.
- splitting reagent must not:
- the AMH N ,c complex may be split before the sample is added to the assay. That way, in the ELISA assay format examples referred to above, the capture antibody will bind to proAMH, as well as AMH N or AMH C , depending on the capture antibody's specificity for AMH.
- the capture antibody is specific for and binds to the N-terminal domain of AMH, then it will bind proAMH and AMH N .
- the capture antibody is specific for and binds to the C-terminal domain of AMH, then it will bind proAMH and AMH C .
- the skilled person will recognise that not all capture antibodies specific for the N- or C-terminal domains of AMH will bind to both proAMH and AMH N or AMH C (e.g. Fig. 6) .
- the proAMH assay according to this aspect of the present invention further comprises:
- preAMH exists in the samples to be analysed
- the assay and methods of the present invention will also detect preAMH as well.
- binding agents and splitting reagents described herein may be used in related methods to determine the level of AMH in a biological sample, and in particular the level of proAMH and AMH N/C .
- a method for measuring the level of proAMH in a subject comprising:
- the splitting reagent splits the AMH N ,c complex into AMH N and AMH C , and wherein when the first binding agent binds to the N-terminal domain of proAMH and AMH N ,c, the second binding agent is specific for and binds to the C-terminal domain of proAMH, or wherein when the first binding agent binds to the C-terminal domain of proAMH and AMH N(C , the second binding agent is specific for and binds to the N-terminal domain of proAMH.
- the AMH N , C assay again uses the non-covalent association between the cleaved AMH species, namely the AMH N(C complex, to distinguish between proAMH and AMH N , C .
- a capture antibody which binds to the C-terminal domain of AMH is bound to a solid support such as, for example, an ELISA plate well, and is used to capture proAMH and AMH N ,c present in a biological sample such as plasma or serum.
- a splitting reagent is then introduced to the assay to split the AMH N ,c complex into AMH N and AMH C .
- the capture antibody only binds to the C-terminal domain of AMH
- the splitting reagent causes dissociation of AMH N , which is subsequently removed by further wash step(s).
- a detection antibody is introduced to the assay which binds to an epitope contained within the C-terminal domain formerly of the AMH N , C complex which epitope was previously shielded by AMH N .
- the detection antibody will not bind to proAMH because the antibody is specific for AMHc, thus providing a specific measure for AMH N(C .
- the antibody specific for AMH C is Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446). This is demonstrated in Fig. 6 where the antibody binds to rhAMHc (lane 2), but not to rhAMH (proAMH ; lanes 3 and 4) under non- reducing/non-denaturing/soluble conditions.
- a person skilled in the art would recognise that the AMH N ,c assay could alternatively be configured using a capture antibody specific for the N-terminal of AMH and a detection antibody specific for AMH N , provided that the detection antibody does not also bind to proAMH .
- a non-antibody detection means could be employed, for example, using a labelled form of AMH N or AMH C .
- the sequence in which these assay steps are performed may be varied.
- the biological sample e.g. plasma or serum
- the splitting reagent such as deoxycholate
- the present invention provides an assay for AMH N(C in a biological sample comprising proAMH and AMH N;C obtained from a su bject, the assay comprising measuring the level of AMH N , C using a first binding agent, a second binding agent and a splitting reagent, wherein the first binding agent binds to proAMH and AMH N(C , the second binding agent binds to AMH N or AMH C but does not bind to proAMH or AMH N , C and wherein the splitting reagent splits the AMH N , C complex into AMH N and AMH C .
- the AMH N , C assay further comprises :
- the level of bound AMH N or AMH C corresponds to the amount of AMH N , C in the sample.
- the AMHN, c assay further comprises:
- the level of bound AMH N or AMH C corresponds to the amount of AMH N(C in the sample.
- a method for measuring the level of AMH N ,c in a subject comprising : (i) obtaining a biological sample from the subject; and
- the first binding agent binds to proAMH and AMH N ,c
- the second binding agent binds to AMH N or AMHc but does not bind to proAMH or AMH N ,c / and wherein the splitting reagent is sufficient to split the AMH N ,c complex into AMH N and AMH C .
- the method further comprises:
- the level of bound AMH N or AMH C corresponds to the amount of AMH N , C in the sample.
- the first binding agent is immobilised on a solid substrate.
- the first binding agent is an antibody or antigen binding fragment thereof which binds to the N-terminal or C-terminal domain of AMH, provided that it binds to both proAMH and AMH N(C -
- the antibody or antigen binding fragment thereof may be selected from the group consisting of a polyclonal, monoclonal, bispecific, chimeric and humanized antibody or an antigen binding fragment thereof.
- the second binding agent comprises a detectable label.
- the second binding agent is an antibody or antigen binding fragment that binds to AMH C or AMH N .
- the antibody or antigen binding fragment thereof may be selected from the group consisting of a polyclonal, monoclonal, bispecific, chimeric and humanized antibody or an antigen binding fragment thereof.
- the second binding agent is Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446).
- the second binding agent is a labelled form of AMH N or AMH C .
- the present invention also contemplates an assay for proAMH comprising a binding agent that is specific for proAMH and does not bind to any other form of AMH, including for example, AMH N ,c / AMH N and AMH C . That is, the binding agent exclusively binds proAMH.
- an assay for proAMH in a biological sample comprising proAMH and AMH N , C obtained from a subject, the assay comprising measuring the level of proAMH in the sample using a binding agent specific for proAMH.
- the assay according to this aspect of the present invention comprises:
- a method for measu ring the level of proAMH in a subject comprising:
- the binding agent is an antibody or an antigen binding fragment, such as, for example, a polyclonal, monoclonal, bispecific, chimeric and humanized antibody or antigen binding fragment thereof.
- a monoclonal antibody or antigen binding fragment thereof is particularly preferred.
- the antibody or antigen binding fragment specific for proAMH binds to an epitope spanning the cleavage site between the N- and C-terminal domains of AMH (i.e. an epitope spanning residues Arg 51 Ser 452 of the human propeptide of AMH).
- the present invention also provides a quantatitive measureme for AMH N , C which is performed using a combination of a conventional assay for AMH and a specific assay for proAMH, as described herein.
- the Applicants demonstrate herein that the total AMH in a biological sample is predominantly comprised of proAMH and AMH N ,c / with no or only trace levels of AMH N or AMH C observed (Figs. 5, 6, 7, 8). Accordingly, using conventional assays to measure the total amount of AMH in combination with an assay specific for proAMH provides the ability to measure AMH NjC .
- the present invention provides an assay for AMH N ,c in a biological sample comprising proAMH and AMH N(C obtained from a subject, the assay comprising :
- the amount of AMH N , C in the sample represents the difference between total AMH and proAMH measured in the sample.
- the present invention also provides a method for measuring the amount of AMH NjC in a subject, the method comprising:
- amou nt of AMH N ,c in the sample represents the difference between total AMH and proAMH measu red in the sample.
- the amount of total AMH in the sample is determined using a conventional assay for AMH, including for example, the Beckman Coulter (Catalogue #A79765) and the ANSH Labs AMH ELISA (Catalogue #A79765).
- a conventional assay for AMH including for example, the Beckman Coulter (Catalogue #A79765) and the ANSH Labs AMH ELISA (Catalogue #A79765).
- the amount of proAMH in the sample is determined using an assay described herein.
- the present invention provides an assay for measuring total AMH in a biological sample comprising proAMH and AMH N , C , the assay comprising :
- the total amount of AMH in the sample represents the sum of the amounts of proAMH and AMH N(C measured in the sample.
- the present invention also provides a method for measu ring total AMH in a biological sample comprising proAMH and AMH N(C , the method comprising :
- the total amount of AMH i n the sample represents the sum of the amounts of proAMH and AMH N , C measured in the sample.
- the amount of proAMH in the sample is determined using an assay or method described herein.
- the amount of AMH N ,c in the sample is determined using an assay or method described herein.
- the present invention also contemplates dual purpose or multi-site assay formats for the detection and measurement of different forms of AMH, including proAMH and AMH N ,c-
- a dual purpose assay or method for detection and measurement of different species of AMH in a biological sample obtained from a subject comprising any combination of the assays or methods described herein.
- the dual purpose assay or method detects and measu res both proAMH and AMH N ,c.
- detection and measurement of the different species of AMH may be achieved simultaneously, that is a signal output for each species of AMH to be detected and measured is generated by the same assay at the same time.
- kits comprising a dual purpose assay or method described herein.
- immunoassays involve contacting a sample containing or suspected of containing a peptide biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of peptides in the sample to the antibody. The signal is then related to the presence or amount of the peptide biomarker in the sample (quantitatively, semi-quantitatively or qualitatively).
- Numerous methods and devices are well known to the skilled artisan for the detection and analysis of peptide biomarkers. For example, United States Patent Nos.
- the assay devices and methods according to the present invention may utilize labelled molecules in various sandwich, competitive, or non-competitive assay formats to generate a signal that is related to the presence or amount of AMH in a sample, including, for example, proAMH and AMH N , C .
- Suitable assay formats used for the present invention include in particular, enzyme-linked immunoassays (ELISA), radioimmunoassays ( IAs), competitive binding assays, and the like.
- ELISA enzyme-linked immunoassays
- IAs radioimmunoassays
- competitive binding assays and the like.
- chromatographic, mass spectrographs, and protein "blotting" methods are also contemplated.
- certain methods and devices, such as biosensors and optical immunoassays may be employed to determine the presence or amount of analytes without the need for a labelled molecule.
- Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in the assays and methods of the present invention.
- Solid supports or phases that may be used to immobilize specific binding agents include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates.
- An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support.
- Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, for example, or by indirect binding. In an example of the latter case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.
- Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein that has affinity for one of the components in the biological system or sample being studied.
- the detectable label is typically conjugated to a binding agent, such as an antibody. Binding of AMH to an antibody to form a complex can be detected directly or indirectly.
- Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
- a detectable reaction product e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.
- a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate
- horseradish peroxidase for example can be incubated with substrates such as o-Phenylenediamine Dihyhydrochloride (OPD) and peroxide to generate a coloured product whose absorbance can be measured, or with luminol and peroxide to give chemiluminescent light which can be measured in a luminometer as is known in the art.
- substrates such as o-Phenylenediamine Dihyhydrochloride (OPD) and peroxide to generate a coloured product whose absorbance can be measured, or with luminol and peroxide to give chemiluminescent light which can be measured in a luminometer as is known in the art.
- Biotin or digoxin can be reacted with binding agents that bind strongly to them.
- the proteins avidin and streptavidin will bind strongly to biotin.
- a further measurable label is then covalently bound or linked thereto either by direct reaction with the protein, or through the use of commonly available
- Detection also includes fluorescence resonance energy transfer (FRET) between fluorescent labels, particularly in dual assay formats according to the present invention for the simultaneous measurement of, for example, proAMH and AMH NiC .
- FRET fluorescence resonance energy transfer
- the present invention also contemplates the analysis of different species of AMH, such as the detection and measurement of proAMH and AMH N , C for example, using multi-site assay formats, as will be known to a person skilled in the art (see for example United States Patent No. 7,541,160).
- Generation of a signal from the label can be performed using various optical, acoustical, and electrochemical methods well known in the art.
- detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc. This list is not meant to be limiting.
- Antibody-based biosensors may also be employed to determine the presence or amount of analytes that optionally eliminate the need for a labelled molecule.
- Immunoassay analysers are also well known and include Beckman Access, Abbott AxSym, Roche ElecSys and Dade Behring Status systems amongst others that are well described.
- Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups.
- Sandwich type assays have greater specificity, speed and greater measuring range.
- an excess of the primary antibody to AMH such as proAMH or AMH N ,c, is attached to the well of an ELISA plate, bead or tube via adsorption, covalent coupling, or a second antibody, as described above for solid phase competition binding assays.
- Sample fluid or extract is contacted with the antibody attached to the solid phase. Because the antibody is in excess this binding reaction is usually rapid.
- a detection antibody to AMH is also incubated with the sample either simultaneously or sequentially with the primary antibody. In certain embodiments, the detection assay is not added to the assay until the AMH N , C complex has been split.
- the detection antibody is chosen to bind to a site on AMH that is different from the binding site of the primary antibody. These two antibody reactions result in a sandwich with the AMH from the sample sandwiched between the two antibodies.
- the detection antibody is usually labelled with a readily measurable compound as detailed above.
- a labelled third antibody that binds specifically to the detection antibody may be contacted with the sample. After washing away the unbound material the bound labelled antibody can be measured and quantified by methods outlined for competitive binding assays,
- various types of immunoassays are used, which may include a competitive type of immunoassay.
- competitive immunoassays include an enzyme immunoassay or enzyme-linked immunosorbent assay (EIA or ELISA), a fluorescent immunoassay, a radiometric or radioimmunoassay (RIA), a magnetic separation assay (MSA), a lateral flow assay, a diffusion immunoassay, an immunoprecipitation assay, an immunosorbent or "antigen-down" assay using an analyte bound to a solid support, or an agglutination assay.
- EIA or ELISA enzyme immunoassay or enzyme-linked immunosorbent assay
- RIA radiometric or radioimmunoassay
- MSA magnetic separation assay
- lateral flow assay a diffusion immunoassay
- an immunoprecipitation assay an immunosorbent or "antigen-down" assay using an analyte bound to a solid support, or an
- a sample contains an unknown amount of analyte to be measu red, which may be a protein such as proAMH or AMH N ,c, or both.
- the analyte may also be termed an antigen.
- the sample may be spiked with a known or fixed amount of labelled analyte.
- the spiked sample is then incubated with an antibody that binds to the analyte, such as proAMH or AMH N(C , so that the analyte in the sample and the labelled analyte added to the sample compete for binding to the available antibody binding sites.
- More or less of the labelled analyte will be able to bind to the antibody binding sites, depending on the relative concentration of the unlabelled analyte present in the sample. Accordingly, when the amount of labelled analyte bound to the antibody is measu red, it is inversely proportional to the amount of unlabelled analyte in the sample. The amount of analyte in the original sample may then be calculated based on the amou nt of labelled analyte measured, usi ng standard techniques known in the art.
- an antibody that binds to the analyte such as proAMH or AMH N ,c / may be coupled with or conjugated to a ligand, wherein the ligand binds to an additional antibody added to the sample.
- a ligand includes fluorescein.
- the additional antibody may be bound to a solid support. The additional antibody binds to the ligand coupled with the antibody that binds in turn to the analyte or alternatively to the labelled analyte, forming a mass complex which allows isolation and measurement of the signal generated by the label coupled with the labelled analyte.
- the analyte to be measured may be bound to a solid support, and incu bated with both an antibody that binds to the analyte and a sample containing the analyte to be measured.
- the antibody binds to either the analyte bound to the solid support or to the analyte in the sample, in relative proportions depending on the concentration of the analyte in the sample.
- the antibody that binds to the analyte bound to the solid support is then bound to another antibody, such as anti-mouse IgG, that is coupled with a label .
- amou nt of signal generated from the label is then detected to measure the amount of antibody that bound to the analyte bound to the solid support. Such a measurement will be inversely proportional to the amount of analyte present in the sam ple.
- Such an assay may be used in a microtiter plate format.
- An additional embodiment of the present invention discloses a method for measuring an amount of AMH, including proAMH or AMH N
- Embodiments of the invention as disclosed herein may be used to perform immunoassays referred to as immunometric, "two-site” or “sandwich” immu noassays, wherein the analyte may be bound to or sandwiched between two antibodies that bind to different epitopes on the analyte, such as proAMH or AMH N(C .
- immunoassays include enzyme immunoassays or enzyme-linked immunosorbent assays (EIA or ELISA), immunoradiometric assays (IRMA), fluorescent immunoassays, lateral flow assays, diffusion immunoassays, immunoprecipitation assays, and magnetic separation assays (MSA).
- a first antibody which may be described as the "capture” antibody, may be bound to a solid support, for which examples have been listed above.
- the capture antibody may be bound to or coated on a solid support using procedures known in the art.
- the capture antibody may be coupled with a ligand that is recognized by an additional antibody that is bound to or coated on a solid support. Binding of the capture antibody to the additional antibody via the ligand then indirectly immobilizes the capture antibody on the solid support.
- An example of such a ligand is fluorescein.
- the second antibody which may be described as the "detection" antibody, may be coupled with a label, which may comprise a chemiluminescent agent, a calorimetric agent, an energy transfer agent, an enzyme, a fluorescent agent or a radioisotope.
- the detection antibody may be coupled with or conjugated with a label using procedures known in the art.
- the label may comprise a first protein such as biotin coupled with the second antibody, and a second protein such as streptavidin that is coupled an enzyme.
- the second protein binds to the first protein.
- the enzyme produces a detectable signal when provided with substrate(s), so that the amount of signal measured corresponds to the amount of second antibody that is bound to the analyte.
- Horseradish peroxidase is an example of such an enzyme; possible substrates include TMB (3,3', 5,5'-tetramethyl benzidine, OPD (o-phenylene diamine), and ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6- sulfonic acid).
- Sandwich immunoassays or sandwich ELISAs are particularly suited for use in the present invention.
- a dipstick type assay may also be used. These assays are well known in the art. They may for example, employ small particles such as gold or coloured latex particles with specific antibodies attached.
- the liquid sample to be measured may be added to one end of a membrane or paper strip preloaded with the particles and allowed to migrate along the strip. Binding of the antigen (such as proAMH and/or AMH N ,c) in the sample to the particles modifies the ability of the particles to bind to trapping sites, which contain binding agents for the particles such as antigens or antibodies, further along the strip. Accumulation of the coloured particles at these sites results in colour development that are dependent on the concentration of competing antigen in the sample.
- the antigen such as proAMH and/or AMH N ,c
- dipstick methods may employ antibodies covalently bound to paper or membrane strips to trap antigen in the sample. Subsequent reactions employing second antibodies coupled to enzymes such as horse radish peroxidase and incubation with substrates to produce colour, fluorescent or chemiluminescent light output will enable quantitation of antigen in the sample.
- a radioimmunoassay may also be used.
- a radiolabeled antigen and unlabelled antigen are employed in competitive binding with an antibody.
- Common radiolabels include 125 I, 131 I, 3 H and 14 C.
- Radioimmunoassays involving precipitation of AMH with a specific antibody and radiolabeled antibody binding protein can measure the amount of labelled antibody in the precipitate as proportional to the amount of the AMH in the sample.
- a labelled AMH species is produced and an unlabelled antibody binding protein is used.
- a biological sample to be tested is then added. The decrease in counts from the labelled AMH is proportional to the amount of AMH in the sample.
- RIA it is also feasible to separate bound AMH from free AMH. This may involve precipitating the AMH/antibody complex with a second antibody. For example, if the AMH/antibody complex contains rabbit antibody then donkey anti-rabbit antibody can be used to precipitate the complex and the amount of label counted. For example in an LKB, Gammamaster counter. See Hunt et al. Clin. Endocrinol. 1997 47 :287-296.
- antibody or antibodies as used herein refers to a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope.
- an immunoglobulin gene or immunoglobulin genes or fragments thereof, capable of specifically binding an antigen or epitope.
- antibody includes antigen binding fragments such as, for example, fragments, subsequences, complementarity determining regions (CDRs) that retain capacity to bind to an antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 : 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
- Single chain antibodies are also included by reference in the term "antibody
- antiserum obtained by immunizing an animal such as a mouse, rat or rabbit with an AMH such as for example, proAMH, AMH N or AMH C , or fragments or antigenic variants thereof.
- an AMH such as for example, proAMH, AMH N or AMH C , or fragments or antigenic variants thereof.
- methods of preparing polyclonal antibodies are known to the skilled artisan.
- Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
- the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
- the immunizing agent may include proAMH, AMH N or AMH C , fragments, antigenic variants thereof or a fusion protein thereof.
- immunogenic proteins include but are not limited to keyhole limpet hemocyanin, bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
- adjuvants examples include Freund's complete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
- the immunization protocol may be selected by one skilled in the art without u ndue experimentation .
- Monoclonal antibodies may be prepared using hybridoma methods well known in the art. For example, refer to Kohler and Milstein (1975) Nature (5517) 256, 495-497), US 4,196,265, US 4,816,567.
- the hybridoma cells may be cultured in a suitable culture medium, alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
- Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the American Type Culture Collection, Virginia, USA. Immunoassays may be used to screen for immortalized cell lines that secrete the antibody of interest. Sequences of proAMH, AMHN or AMH C or fragments or antigenic variants thereof may be used in screening.
- Well known means for establishing binding specificity of monoclonal antibodies produced by the hybridoma cells include immunoprecipitation, radiolinked immunoassay ( IA), enzyme-linked immunoabsorbent assay (ELISA) and Western blot. (Lutz et al. (1988) Exp. Cell. Res. 175 : 109-124).
- IA radiolinked immunoassay
- ELISA enzyme-linked immunoabsorbent assay
- Western blot Western blot.
- the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Mu nson et al. (1980) Anal Biochem 107: 220. Samples from immunised animals may similarly be screened for the presence of polyclonal antibodies.
- Monoclonal antibodies can also be obtained from recombinant host cells.
- DNA encoding the antibody can be obtained from a hybridoma cell line.
- the DNA is then placed into an expression vector, transfected into host cells (e.g., COS cells, CHO cells, E. coll cells) and the antibody produced in the host cells.
- the antibody may then be isolated and/or pu rified using standard techniques.
- the monoclonal antibodies or fragments may also be produced by recombinant DNA means (see for example US 4,816,567). DNA modifications such as substituting the coding sequence for hu man heavy and light chain constant domains in place of the homologous mu rine sequences (US 4,816,567 above) are also possible.
- the antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art (see, for example, United States Patent Nos. 5,334,708, 5,821,047, and 7,476,724) . Production of chimeric (US 4,816,567), bivalent antibodies (US 5,843,708) and multivalent antibodies are also contemplated herein (US 6,020,153).
- the monoclonal antibodies secreted by the cells may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, reverse phase HPLC, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. See, for example, Scopes, Protein Purification : Principles and Practice, Springer-Verlag, NY (1982).
- Bispecific antibodies may also be useful. These antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Antibodies with greater than two specificities for example trispecific antibodies are also contemplated herein.
- Antibodies used in the immunoassays described herein preferably specifically bind to AMH, such as proAMH or AMH N;C .
- AMH such as proAMH or AMH N;C .
- the term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s).
- the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule.
- antibodies bind with affinities of at least about "6 , or 10 "7 M, or at least about 10 ⁇ 8 M, or 10 "9 M, or 10 "10 , or 10 "11 or 10 "12 M.
- phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte
- a basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome that encodes the polypeptide.
- polypeptides The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target binds to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. For example, refer to US 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.
- the antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding.
- the screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h.
- microtiter wells are then washed and a labelled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a colour reaction will appear where antibody to the immobilized polypeptide(s) is present.
- a labelled secondary antibody for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies
- the antibodies so identified may then be further analysed for affinity and specificity in the assay design selected.
- the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
- kits for performing the assays and methods described herein.
- Suitable kits comprise reagents sufficient for performing an assay for at least one of the described AMH species, together with instructions for performing the described threshold comparisons.
- kits will be formatted for assays known in the art, and in particular, ELISA assays.
- kits for measuring proAMH in a biological sample obtained from a subject comprising proAMH and AMH N ,c, the kit comprising a first binding agent, a second binding agent, a splitting reagent and instructions for how to measure the level of proAMH in the sample.
- the components of the kit are further characterised :
- the splitting reagent is capable of splitting the A H N , C complex into AMH N and
- AMH N(C ) the second binding agent is specific for and binds to the N-terminal domain of proAMH.
- kits for measuring AMH N ,c in a biological sample comprising proAMH and AMH N , C obtained from a subject, the kit comprising a first binding agent, a second binding agent, a splitting reagent, and instructions for how to measure the level of AMH N ,c in the sample.
- the components of the kit are further characterised :
- the splitting reagent splits the AMH N , C complex into AMH N and AMH C ; and (ii) when the first binding agent binds to proAMH and AMH N
- reagents for performing such assays are provided in an assay device, and such assay devices may be included in such a kit.
- preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising an antibody that detects the AMH species bound to a solid support.
- the first binding agent is immobilised on a solid support.
- such reagents can also include one or more detectably labelled antibodies, the detectably labelled antibody comprising an antibody that detects the intended AMH species bound to a detectable label. Additional optional elements that may be provided as part of an assay device are described hereinafter.
- Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, electrochemical luminescence (eel) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indi rectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through the use of a specific binding molecule which itself may be detectable (e.g., a labelled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.) .
- the second binding agent comprises a detectable label.
- kits for measuring the level of proAMH in a biological sample comprising proAMH and AMH N , C obtained from a subject, the kit comprising a binding agent specific for proAMH.
- the binding agent specific for proAMH binds to an epitope that spans the cleavage site between the N- and C-terminal domains of proAMH.
- the binding agents comprised within the kits of the present invention may include an antibody or an antigen binding fragment thereof, for example, a monoclonal antibody or antigen binding fragment thereof.
- an antibody or an antigen binding fragment thereof for example, a monoclonal antibody or antigen binding fragment thereof.
- the second binding agent may be a labelled form of AMH N or AMH C .
- the splitting reagent of the kits described herein comprises deoxycholate.
- kits for the analysis of AMH including proAMH and AMH N , C .
- the kit comprises reagents for the analysis of at least one test sample.
- the kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein.
- Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labelled species for performing a competitive assay, for an analyte, such as proAMH or AMH N>C .
- an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies will bind different forms of AMH, such as proAMH or AMH N , C .
- each of the antibodies used in the kits of the present invention include monoclonal antibodies.
- the instructions for use of the kit and performing the correlations can be in the form of labelling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use.
- labelling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.
- kits comprising dual purpose or multi-site assays for the detection and measurement of different species of AMH, including proAMH and AMH N , C . That is, the present invention provides assays and kits capable of simultaneously determining the presence and amount of different species of AMH, such as AMH N)C and proAMH, in a biological sample which has been obtained from a subject. In certain embodiments, the present invention provides dual purpose assays and kits comprising dual purpose assays for the simultaneous measurement of proAMH and AMH N(C , wherein the assay comprises any combination of the assays described herein.
- Example 12 including Tables 3-5 and in Figures 23 and 24.
- proAMH levels can be measured in human serum from healthy individuals recruited from the community,
- the levels of proAMH, total AMH and %proAMH vary between individuals, and between distinct population groups; and
- proAMH ELISA described herein generates data that is distinct from the existing commercially available AMH ELISAs.
- the assays, methods and kits described herein are useful in the measurement of different forms of AMH present in a biological sample, for example serum. This information can be used to differentiate the biological functions of the various AMH species.
- Recombinant human AMH was produced from HEK293 cells transfected with a proAMH vector (PxTherapeutics SA (Grenoble, France), under contract to the University of Otago) .
- the resultant rhAMH was a mixture of proAMH, with lesser amounts of AMH N(C (Fig. 3) .
- Recombinant human AMH N was produced from HEK293 cells transfected with a AMH N vector (PxTherapeutics SA (Grenoble, France), under contract to the University of Otago) .
- a solution of enriched in rhAMHN, C and diminished in proAMH was manufactured by cleaving batches of the rhAMH (PxTherapeutics) with fu rin. The following conditions produce extensive cleavage, producing a solution with only trace levels of proAMH .
- 10 nM rhAMH was reacted with 8 units of fu rin (New England Biolabs) in 40 pL of reaction buffer consisting of 100 mM HEPES, pH 7.4, 1.8 mM Ca 2+ , 140 mM Na + , 4 mM K + , 0.05% v/v Tween-20 for 24 hours at 37°C.
- rhAMH was purchased from R&D systems (1737- MS) .
- the anti-N terminal antibody was purchased from R&D systems (Catalogue* AF2748, anti-human AMH propeptide antibody) . Epitopes from the C-terminus to the cleavage site of AMH were detected or precipitated with a either Goat anti-human AMH/MIS antibody (R&D systems, Catalogue* AF1737) (Fig. 5) and Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446) (Fig. 6) . IRDye680 Donkey anti-Goat IgG antibody (Licor) was used as the secondary antibody for western blot.
- IRDye680 Donkey anti-Goat IgG antibody (Licor) was applied at 0.66-1 yg/mL for 10-60 minutes before final rinsing and drying of the blotting membrane (Figs 4, 5, 7- 10).
- the blotting membrane was imaged using the Odyssey fluorescent infrared scanner (Licor).
- the voltage (gain) to the photomultiplier tube was used to vary the intensity of the blot depending on the signal strength of the relevant bands.
- the grayscale has been inverted to present the image as black-on-white images.
- the density of the bands in some of the western blots were quantified without image manipulation, using Image J software (NIH, http://rsbweb.nih.gov/ij7).
- the original image was then enhanced linearly to give visual confirmation of the densitometry measurements.
- This involved a linear stretch of the dynamic range of the image intensity such that the range of pixel intensities occupied by the histogram was linearly recalculated to occupy the full 256 bit-space with image J.
- the "dust and speckles" filter in Photoshop CS5 (Adobe) with a pixel radius of 6 and a threshold of 10 (image resolution : 300 pixels/inch) was used to reduce the auto-fluorescence of dust particles in the image.
- Plasma Blood samples were collected in EDTA coated tubes (BD) and the plasma obtained by centrifugation at 2 000 xg for 5 minutes. Serum: 2 mL of blood was collected into BD vacutainers by either a registered phlebotomist or licenced medical practioner. The blood sat room temperature for between 30 minutes and 1 hour, after which the serum was removed, divided into aliquots, snap frozen and stored at -80°C. Again, the sex, age and blood AMH concentration of each participant is recorded in Table 1.
- AMH was immunoprecipitated with either polyclonal anti-human proAMH goat IgG (R&D systems, AF2748, amino acids 19-450) or a mixture of polyconal antibodies to the C- terminal peptide (amino acids 447-553): anti-human AMH/MIS goat IgG and and goat anti- rat AMH/MIS antibody (R&D systems, AF1446, AF2748) (Figs 3-6).
- the primary antibody was cross-linked to the beads for the analysis of the gycosylation state of AMH to minimize the breadth of the IgG band (Fig. 8 Figs. 9, 10).
- Rabbit anti-goat IgG Trueblot beads were incubated with goat anti-human AMH propeptide antibody for 1 hour at room temperature with agitation.
- the beads were rinsed twice with 0.01 M PB and twice with 0.2 M triethanolamine (BDH), pH 8.2.
- BDH triethanolamine
- the associated proteins were cross-linked with 25 mM dimethyl pimelidate dihydrochloride (Sigma), 0.2 M triethanolamine, pH 8.2 for 45 minutes at room temperature.
- the reaction was stopped with one quick rinse, followed by 1 hour of incubation in 0.1 M ethanolamine (Sigma), pH 8.2.
- the beads were rinsed 2 times with 0.01 M PB and unbound antibody was eluted with 0.1 M glycine-HCI (BDH), pH 2.5 followed by 2 rinses in 0.01 M
- the plasma of boy B3 was diluted 1 :2 with 0.01 M PB.
- the AMH was immunoprecipitated from the sample with 1 pg of goat anti-human AMH propeptide antibody cross-linked to rabbit anti-goat IgG Trueblot beads. Incubation was conducted overnight at 37°C with 0.03% sodium azide (Merck), 1 ⁇ phenylmenthanesulfonyifluoride (Sigma) and complete mini protease inhibitor cocktail (Roche) diluted as per manufacturer's instruction.
- the beads were then rinsed twice with 1 ml_ 0.01 M PB resuspended in 1 mL 0.01 M PB and transferred to a new microtube.
- Enzymatic deglycosylation was conducted using the EDEGLY enzymatic deglycosylation kit (Sigma). To elute the precipitated AMH, the beads were pelleted, the supernatant was removed and 60 pL of ultra-pure water, 25 ⁇ _ of the 5x reaction buffer and 2.2 pL of 5% SDS (Sigma) solution was added. The sample was heated to 95°C for 5 minutes and the supernatant was transferred to new tubes in 21 pL aliquots. Disulfide bonds in the samples were reduced with 0.9 pL of 2-mercaptoethanol and heating to 95°C for 5 minutes.
- the AMH concentration in the plasma and serum samples and the rhAMH were assayed using the Beckman Coulter AMH Generation II ELISA Kit (Beckman Coulter). Standard curves for interpolation were generated by fitting a quadratic curve to the absorbance values obtained for the AMH standard solutions (Beckman Coulter).
- Serum or recombinant proteins were added to the ELISA wells, typically with a volume of 20 pL. 100 pL of the manufacturer's assay buffer was immediately added. The wells were incubated for 1 hour at room temperature on an orbital shaker at 300 rpm. The wells were washed 10 times with 400 ⁇ _ of ELISA wash buffer, using a ELISA plate washer. The wash buffer was removed and 40 ⁇ _ of Laemmli buffer added to each well and covered with a pre-cut square of 96-well plate adhesive cover. The wells were incubated on a metal plate at 95°C for 6 minutes, after which the wells were cooled on the bench.
- Samples containing AMH were applied to a Beckman Coulter (BC) AMH Gen II ELISA plate at a volume of 20 ⁇ 1_. Assay buffer was then added to the ELISA plate wells (100 pL) and the samples were incubated at room temperature for 1 hour on an orbital shaker at 300 rpm. The wells were washed 5 times with the ELISA kit wash buffer. Two changes of 150 pL of 0.2% sodium deoxycholate in 0.02 M Phosphate buffer pH 6.8, pre-warmed to 37°C were applied to the wells and were incubated for 15 minutes each at 37°C. The deoxycholate is used to dissociate the AMH N(C complex without affecting overall binding of the AMH species to the ELISA plate.
- BC Beckman Coulter
- Assay buffer was then added to the ELISA plate wells (100 pL) and the samples were incubated at room temperature for 1 hour on an orbital shaker at 300 rpm. The wells were washed
- the wells were washed 5 times with the ELISA kit wash buffer. 100 pL of AMH Gen II kit biotinylated detection antibody solution was applied then the plate was incubated at room temperature for 1 hour on an orbital shaker at 300 rpm. The wells were then washed 5 times with the ELISA kit wash buffer. Streptavidin- horse radish peroxidase conjugate was applied at 100 pL to the ELISA wells and was incubated at room temperature for 30 minutes on an orbital shaker at 300 rpm. The wells were washed 5 times with the ELISA kit wash buffer.
- TMB was applied at 100 pL to all wells and was incubated at room temperature for 8-12 minutes, after which 100 pL of stop- solution was applied. Absorbance was read at 450 nm and 570 nm for background correction. The 570 nm absorbance was subtracted from that at 450 nm to obtain the true absorbance reading.
- AMH N ,c ELISA AMH N ,c ELISA
- the AMH-Duoset capture antibody (R&D systems, catalogue #DY1737) was diluted to 4 pg/ml in 0.05 M Carbonate buffer pH 9.6-9.8 and 100 pL was applied to a 96-well plate 18 hours incubation at 4°C. The plate was washed 3 times with phosphate-buffered saline, pH 7.4 with 0.05% Tween 20 (Sigma, catalogue#P-1739) (PBS-T). The wells were incubated for 30 minutes with 300 pL of phosphate-buffered saline, pH 7.4, 1% Tween 20 at room temperature.
- the plate was washed 3 times with PBS-T and 100 pL of AMH-Duoset detection antibody (R&D systems, catalogue #DY1737), diluted to 2 pg/mL in 1% BSA in PBS-T was applied to the wells and was incubated at room temperature for 1 hour.
- the plate was washed 3 times with PBS-T and 100 pL of Amersham Streptavidin-Biotinylated Horseradish Peroxidase Complex (GE healthcare, catalogue#RPN1051) diluted 1 :2000 in PBS-T was applied for 15 minutes at room temperatu re.
- the plate was washed 3 times with PBS-T.
- TMB (KPL, catalogue#50-76-l l) was applied at 100 pL to all wells and was incubated at room temperature for 15-20 minutes, after which 100 pL of 2M sulphuric acid was applied .
- Absorbance was read at 450 nm and 570 nm for background correction. The 570 nm absorbance was subtracted from that at 450 nm to obtain the true absorbance reading.
- Example 2 Validation of AMH antibodies for western blotting
- the molecular weights and immunoreactivity of proAMH, AMH N , AMH C and AMH255-560 in a reducing-conditions western blot were examined using antibodies to the N- and C- terminal portions of AMH (Fig. 3).
- the rhAMH derived from HEK cells was predominantly a 72 kDa species, with lesser amounts of other forms of AMH.
- the 72 kDa species is designated proAMH as it bound both the C- and N-terminal antibodies, and because pretreatment with the proprotein convertase furin diminished the intensity of the band.
- AMHc at 12.5 kDa was detected exclusively by the C-terminal antibody, whereas two AMH N bands at 62 and 64 kDa were detected exclusively by the N-terminal antibody.
- AMH C and AMH N bands increased following furin treatment, providing further confirmation of their identities.
- a lower abundance 47 kDa band was also present.
- This band contained both N and C-terminal epitopes, with its size being consistent with the previously described AMH 255- 56o.
- AMH in blood is too low to be directly analyzed by western blots, necessitating prior concentration by immunoprecipitation.
- Applicants therefore first established that the non-specific bands associated with immunoprecipitation were distinct from the main AMH species.
- the proteins immunoprecipitated from human serum by a control goat IgG were different in size to proAMH, AMH N and AMH C (Fig. 4). Faint nonspecific bands were detected at approximately 47 kDa. These bands would not obscure the presence of significant levels of AMH 255 - 56 o.
- Example 3 AMH N C and ProAMH is present in human blood
- the blood of a boy was then examined by separate immunoprecipitation with anti-N- or anti-C-terminal antibodies to AMH, followed by separate detection with the anti-N- and anti-C-terminal antibodies. Significant levels of both proAMH and A H N , C were detected.
- the strongest band immunoprecipitated by the N-terminal a ntibody had the characteristics of proAMH . That is, a reduced 72kDa protein that reacts with both the anti-C and anti-N terminal antibodies (Fig . 5A,B) . This band was not detected in the blot of a post- menopausal woman (Fig . 5A,B), who only had trace levels of AMH (Table 1).
- the N-terminal antibody also immunoprecipitated bands with molecular weights appropriate for AMH N (Fig. 5A) and AMHc (Fig. 5B). Neither of these bands were present in the negative control (post-menopausal woman).
- the detection of AMH C suggests it is co- immunoprecipitating as part of the AMH N , C complex as the antibody to the N-terminal domain does not directly bind to AMH C (Fig. 5A).
- the C-terminal antibody immunoprecipitates rhAMH c but does not precipitate protein from the rhAMH (Fig. 6), which is a mix of proAMH and AMH N , C (Fig. 3).
- This antibody precipitated trace levels of AMH N from the blood of the boy, with no detectable levels of either AMH C or proAMH. This indicates that the boy's blood contained little or no free AMH C , providing independent validation that the AMH C precipitated by the anti-N antibody is due to the presence of AMH N , C in blood.
- the trace AMH N band suggests that the anti-C terminal antibody inefficiently precipitates ⁇ , ⁇ with the greater sensitivity of the N- than the C- terminal detection permitting the visualisation of the precipitated AMH N .
- the anti-N terminal was used for immunoprecipitation, with a combination of the anti-N and anti-C terminal antibodies used for the western blots to ensure that all precipitated species were detected.
- Example 4 proAMH and AMH N , C is present in adult serum, from both sexes
- AMH was immunoprecipitated from plasma samples from 2 additional pre-pu bescent boys (B2, B3), 3 young men (Ml, M2, M3) and 3 pre-menopausal women (Wl, W2, W3), with a post-menopausal woman included as a control (Fig. 7).
- ProAMH and AMH N were detected in the blood of the boys, men and women (Fig . 7) with the intensity of the bands being broadly in line with the ELISA estimate of AMH levels (Fig. 7, Table 1) .
- the postmenopausal woman had no detectable levels of AMH N , and only trace levels of proAMH.
- the anti-N terminal antibody co-immunoprecipitated AMH C from the serum of boys.
- the density of the AMH C bands in the adult samples were low as expected (Fig . 7 & 8A).
- the presence of AMH C was therefore confirmed by densitometry (Fig. 8C), after which the visibility of the bands was made clearer by linear contrast enhancement (Fig. 8B) .
- Table 1 Study participant's age, sex and blood AMH concentration
- the rhAMH had two AMH N bands (Fig. 3), with some of the AMH N bands precipitated from human blood having apparent minor variation in molecular weight.
- AMH has two putative N-linked glycosylation sites in the N-terminal region (Cate et al. (1986) Cell 45:685-698), which may account for this. Applicants therefore examined the glycosylation status of AMH in human blood, by investigating whether deglycosylation enzymes reduced the molecular weight of the proAMH and/or the AMH N bands.
- a deglycosylation kit (Sigma Catalogue # E-DEGLY) was used to remove all N- and O-linked glycans This reduced the size of the proAMH and AMH N bands, yielding several lower molecular weight species (Fig. 9).
- AMH species (Fig. 3)
- recombinant human AMH C (rhAMH c )
- recombinant human AMH N ( ⁇ ) were added to separate ELISA wells.
- the wells were then washed using standard techniques according to the manufacturers instructions to remove any non-specifically bound AMH species.
- the captured proteins were then analyzed by western blot which included the recombinant proteins as a control (Fig. 11) .
- the BC ELISA plate bound proAMH, as evidenced by an appropriately sized band that was detected by both N-terminal and C-terminal antibodies to AMH (Figs. 11A and 11B). This is an essential property of the capture antibody.
- the BC ELISA matrix also captured AMH C from the rhAMH c preparation; AMH C , AMH N , and AMH 25 5-56o from rhAMH.
- the ELISA plate did not capture rhAMH N from the rhAMH N preparation.
- the pattern of capture is consistent with the capture antibody being either an antibody to the C-terminal region (with the AMH N being captured as part of AMH N , C ) or an antibody that recognises an epitope involving amino acids present on both AMH N and AMH C .
- C is not an essential feature of the proAMH ELISA, but is used here to demonstrate that the capture of AMH N , C does not intefere with the measurement of proAMH .
- the AMH ELISA plate captured proAMH, AMH C and AMH N from the blood of boys as evidenced by western blot analysis (Fig. 12).
- the western blot staining pattern observed for the serum sample obtained from boys was identical to that observed for the recombinant forms of AMH (Fig. 11), except that no AMH 2 55-5 6 o was present.
- rhAMHc, rhAMH N , rhAMH or human serum were loaded onto wells from the Ansh Labs assay.
- the captured proteins were then recovered an anlayzed by western blots using a detection antibody to either the N-terminal domain or C-terminal of AMH .
- the proAMH, AMHc, AMHN and AMH255-560 bands are indicated on the western blot in Fig . 17, confirming that N-terminal capture will achieve the same outcome.
- Example 7 Splitting of AMH N , C complex
- the AMH Ni c complex may be split before addition of the sample to the assay matrix or after the AMH species have been captured.
- AMH N C complex is split before addition to the ELISA matrix
- Recombinant human AMH was treated with furin.
- Recombinant human AMH at 10 nM was incubated with 8 units of furin in 100 mM HEPES, pH 7.4, 1.8 mM Ca 2+ , 140 mM Na + , 4 mM K + , 0.05% v/v Tween-20.
- the reaction was allowed to proceed at 37°C for 24 hours to produce the cleaved form of AMH (rhAMH N(C ), with only trace levels of proAMH present.
- Varying concentrations of sodium deoxycholate at 0.05%, 0.1% or 0.2% w/v were added to rhAMH or rhAMH N ,c samples, and the samples incubated at 37°C. Following deoxycholate treatment, the samples were then added to an ELISA plate (Beckman Coulter; Catalogue # A79765).
- Serum was added to a total of six wells, and three of the wells were then washed twice with 0.2 % deoxycholate, prewarmed to 37°C for 15 minutes at 37°C per wash.
- the deoxycholate wash released the captured AMH N , without loss of the captured proAMH, as evidenced by western blot (Fig. 14).
- a higher concentration of deoxycholate (0.2%) was used to split the AMH N ,c complex when bound to the ELISA plate, compared to when the AMH N , C complex is split prior to its capture (example above).
- the required concentration of the spitting reagent will be affected by temperature, time and context (i.e. the sample to which it is added).
- the optimal concentrations of splitting reagent for splitting prior to addition to the ELISA plate, and after the capture step need to be separately determined.
- Applicants modified existing conventional AMH ELISAs to include a deoxycholate wash step following protein capture, namely, the Beckman Coulter (BC ELISA; Catalogue #A79765) and the ANSH Labs AMH ELISA (ANSH ELISA; Catalogue #A79765). As shown below, this modification changed the ELISA from an assay specific for the sum of proAMH + AMH NiC , to an assay which is specific for proAMH.
- BC ELISA Beckman Coulter
- ANSH ELISA ANSH Labs AMH ELISA
- the BC ELISA did not detect rhAMH c and rhAMH N , which in combination with the above observation shows that the detection and capture antibodies recognise epitopes on different termini of AMH (i.e. N-terminal and C-terminal domains).
- Example 9 proAMH ELISA detects proAMH in human samples
- Serial dilution of serum AMH shows a parallel relationship to serial dilution of the rhproAMH standard in the proAMH ELISA.
- Serum from a human male underwent 1: 1 serial dilution in an identical manner to the standard curve.
- the concentration of the undiluted human serum was interpolated from the standard curve and the concentrations of AMH in the diluted serum were calculated using the dilution factor.
- Fig. 18 shows that the ELISA detects AMH from human samples and there is no difference in detection compared to the rhproAMH used for the standard.
- Example 10 AMH N , C specific ELISA
- Applicants will modifiy existing ELISA formats, for example the Beckman Coulter assay (Catalogue #A79765), to include a deoxycholate wash step either before or after protein capture. This modification will change the ELISA from an assay specific for the sum of proAMH + AMH N , C , to an assay which is specific for AMH N ,c.
- the assay will employ a detection antibody which specifically binds to AMH C but not to proAMH.
- the detection antibody Goat anti-rat AMH/MIS antibody (R&D systems, Catalogue* AF1446)
- rhAMH c lane 2
- proAMH proAMH
- the ELISA will be performed under similar conditions (e.g. non- reducing/non-denaturing/soluble conditions very similar to the immunoprecipitation conditions used in Fig. 6)
- the detection antibody will bind to and detect only AMH C .
- the amount of bound AMH C will be directly proportional to the amount of AMH N ,c (i.e. that which existed prior to the splitting step).
- AMH duoset ELISA detects AMH C with high efficiency, although it does not detect proAMH and the signal is greatly diminished for AMH NjC (Fig. 19).
- AMHc i.e. not proAMH or AMH N , can be recovered from the multi-well plate.
- proAMH and AMH N , C are sterically hindered from binding to the capture antibody.
- the weak detection of proAMH is explained by the sample containing small quantities of AMH N ,c- Similarly, the weak detection of AMH NjC must occur due to some degree of dissociation and reassociation between AMH N and AMH C in the AMH N ,c complex.
- C complex dissociates allowing the AMHc fragment to bind to the ELISA capture antibody.
- Free-AMH C is not known to exist in human serum and therefore any AMH detected by this ELISA after deoxycholate pre- treatment of serum samples will be AMH N ,c and not proAMH.
- Example 12 Relative abundance of proAMH and AMH N(C in the blood of healthy individuals Applicants sought to describe the relative abundance of proAMH and AMH NiC in the blood of healthy individuals recruited from the community. The study incorporates a comparison between the sexes and between the major stages of the life cycle for each sex.
- the male dataset contained : 131 boys aged from 4 to 11 years old; 100 men aged between 24 and 65 years; 101 elderly men, who were older than 65 years (Table 3).
- the female dataset contained: 20 girls aged from 4 to 11 years old; 34 women aged between 19 and 31 years old and 25 women aged between 31 and 46 years old (Table 3).
- the stratification at the age of 31 years delineates the ages of peak and diminishing fertility (http://www.ncbi.nlm.nih.gov/pubmed/19589949).
- Elderly women were not examined as they lack detectable levels of total AMH (Chong et al. (2012) Int J Gynecol Cancer 22: 1497- 1499) .
- Total AMH was measured using Beckman Coulter's AMH Gen II ELISA and standards, according to the manufacturer's instructions, in accordance with field safety notice FSN- 20434-3, June 2013.
- the serum of boys was diluted 1 :9 in sample diluent before assay.
- ProAMH was quantified with the proAMH assay as described in Examples 8 and 9, using the AMH Gen II ELISA plate, detection reagents and ELISA standards.
- the proportion of total AMH that was proAMH was calculated as defined above. When a ratio of two numbers close to zero is taken, the resulting number is very sensitive to any imprecision in the original numbers, particularly the denominator. ELISA values near the limit of detection have elevated error. For this reason, the %proAMH values were not calculated when an individual's total AMH levels were less than 10 pM. The following number of participants were excluded by this criteria: 0 boys; 0 younger men; 6 elderly men; 3 girls; 3 younger women and 13 women from the 31-46 age group.
- proAMH levels can be measured in human serum
- proAMH levels of proAMH, total AMH and %proAMH vary between individuals, and between distinct population groups.
- the proAMH ELISA generates data that is distinct from the existing AMH E LISAS
- the level of proAMH in boys was invariably high, but with an inter-person variation that spanned an order of magnitude.
- the minimum level of proAMH in the 131 boys was 74.9 pM, a value that was over double the maximum value in all other population groups (Table 4).
- %proAMH in boys exhibited biological variation, with a mean of 33% and a standard deviation of 4.4% (Table 5). The ranges of %proAMH in the boys and the men samples did not overlap, indicating that the relative levels of proAMH and AMH NiC change markedly during the pubescent transition.
- the absolute levels of proAMH and the %proAMH in the samples from men were significantly lower than those for boys (Tables 4, 5) .
- the absolute level of proAMH was variable between men, with a range of 2.1 to 33.5 pM. This range has extensive overlap with that the range for women .
- the %proAMH in men tended, however, to be lower than in women.
- proAMH proAMH in young women were little different to those of girls. All women had detectable levels of proAMH, but the women in the 31-46-year-old group had significantly less proAMH, on average. This contrasts with the women's level of total AMH which were higher in young women than in girls (Table 4) .
- the %proAMH range in the young women was lower and narrower than that of the girls. Older women had a higher mean %proAMH value than younger women, with the range of %proAMH levels trending towards that of girls (Table 5) .
- any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms in the specification.
- the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation.
- the assays and methods illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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US14/900,581 US20160370385A1 (en) | 2013-06-21 | 2014-06-20 | Assay for anti-mullerian hormone |
EP14813075.0A EP3011337A4 (en) | 2013-06-21 | 2014-06-20 | Assay for anti-mullerian hormone |
AU2014281270A AU2014281270A1 (en) | 2013-06-21 | 2014-06-20 | Assay for Anti-Mullerian Hormone |
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US201361838053P | 2013-06-21 | 2013-06-21 | |
US61/838,053 | 2013-06-21 | ||
US201361871025P | 2013-08-28 | 2013-08-28 | |
US61/871,025 | 2013-08-28 |
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PCT/NZ2014/000123 WO2014204327A1 (en) | 2013-06-21 | 2014-06-20 | Assay for anti-mullerian hormone |
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US (1) | US20160370385A1 (en) |
EP (1) | EP3011337A4 (en) |
AU (1) | AU2014281270A1 (en) |
WO (1) | WO2014204327A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3257867A1 (en) * | 2016-06-17 | 2017-12-20 | Biomérieux | Method for preparing anti-amh antibodies and uses thereof |
CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
WO2020103691A1 (en) | 2018-11-20 | 2020-05-28 | 厦门万泰凯瑞生物技术有限公司 | Specific antibody for amh, and uses thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111999509A (en) * | 2020-08-10 | 2020-11-27 | 深圳市宇诺生物技术有限公司 | Anti-mullerian hormone determination kit, preparation method and detection method |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989006695A1 (en) * | 1985-10-30 | 1989-07-27 | Biogen, Inc. | Cleaved dimers of mullerian inhibiting substance-like polypeptides |
WO2006127850A1 (en) * | 2005-05-24 | 2006-11-30 | Beckman Coulter, Inc. | Immunological assays and antibodies for anti-mullerian hormone |
EP2161579A1 (en) * | 2008-08-26 | 2010-03-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Detection method of biologically active forms of anti-mullerian hormone |
WO2014074835A2 (en) * | 2012-11-09 | 2014-05-15 | Ansh Labs Llc | Antibody compositions and immunoassay methods to detect isoforms of anti-mullerian hormone |
-
2014
- 2014-06-20 EP EP14813075.0A patent/EP3011337A4/en not_active Withdrawn
- 2014-06-20 AU AU2014281270A patent/AU2014281270A1/en not_active Abandoned
- 2014-06-20 WO PCT/NZ2014/000123 patent/WO2014204327A1/en active Application Filing
- 2014-06-20 US US14/900,581 patent/US20160370385A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989006695A1 (en) * | 1985-10-30 | 1989-07-27 | Biogen, Inc. | Cleaved dimers of mullerian inhibiting substance-like polypeptides |
WO2006127850A1 (en) * | 2005-05-24 | 2006-11-30 | Beckman Coulter, Inc. | Immunological assays and antibodies for anti-mullerian hormone |
EP2161579A1 (en) * | 2008-08-26 | 2010-03-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Detection method of biologically active forms of anti-mullerian hormone |
WO2014074835A2 (en) * | 2012-11-09 | 2014-05-15 | Ansh Labs Llc | Antibody compositions and immunoassay methods to detect isoforms of anti-mullerian hormone |
Non-Patent Citations (4)
Title |
---|
DI CLEMENTE, N. ET AL.: "Processing of Anti-Müllerian Hormone Regulates Receptor Activation by a Mechanism Distinct from TGF-beta", MOLECULAR ENDOCRINOLOGY, vol. 24, no. 11, 2010, pages 2193 - 2206, XP055108341 * |
KUMAR, A. ET AL.: "Development of a second generation anti-Müllerian hormone (AMH) ELISA", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 362, 2010, pages 51 - 59, XP027509590 * |
LONG, W.-Q. ET AL.: "Detection of Minimal Levels of Serum Anti-Müllerian Hormone during Follow-Up of Patients with Ovarian Granulosa Cell Tumor by Means of a Highly Sensitive Enzyme-Linked Immunosorbent Assay", THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 85, no. 2, 2000, pages 540 - 544, XP002481798 * |
See also references of EP3011337A4 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3257867A1 (en) * | 2016-06-17 | 2017-12-20 | Biomérieux | Method for preparing anti-amh antibodies and uses thereof |
WO2017216334A1 (en) * | 2016-06-17 | 2017-12-21 | bioMérieux | Method for preparing anti-amh antibodies and uses of same |
CN109563163A (en) * | 2016-06-17 | 2019-04-02 | 生物梅里埃公司 | It is used to prepare the method and application thereof of anti-AMH antibody |
RU2764198C2 (en) * | 2016-06-17 | 2022-01-14 | Биомерье | Method for producing antibodies against amh and their application |
US11225518B2 (en) | 2016-06-17 | 2022-01-18 | bioMérieux | Method for preparing anti-AMH antibodies and uses of same |
CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
WO2020103691A1 (en) | 2018-11-20 | 2020-05-28 | 厦门万泰凯瑞生物技术有限公司 | Specific antibody for amh, and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
AU2014281270A1 (en) | 2016-01-21 |
EP3011337A4 (en) | 2016-12-21 |
US20160370385A1 (en) | 2016-12-22 |
EP3011337A1 (en) | 2016-04-27 |
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