A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone

• Blood contains uncleaved precursor (proAMH) and receptor-competent AMH (AMHN,C).
• ProAMH and AMHN,C are not readily distinguished by immunoassays.
• The addition of sodium deoxycholate generates a specific proAMH assay.
• The assay works by dissociating AMHN,C which is a noncovalent complex.
• The assay does not require cleavage-state-specific antibodies.

The utility of serum anti-Müllerian hormone (AMH) assays in assessment of female fertility have been investigated extensively but little is known about the biological activity of the hormone being studied. ProAMH is the proprotein precursor and is incapable of binding to the AMH-specific type II receptor. Proteolytic cleavage generates receptor-competent AMHN,C which is a non-covalent complex of the N- and C-terminal cleavage fragments. Commercially available AMH assays do not differentiate the two forms of AMH. Techniques were developed to dissociate the AMHN,C complex and abolish its two-site immunoassay immunoreactivity. This allowed specific quantification of proAMH. The surfactant sodium deoxycholate (DOC) dissociated AMHN,C without disrupting binding of proAMH to the capture-antibody with an optimal concentration of 0.1–0.2%w/v. The incorporation of a DOC incubation step into the AMH Gen II ELISA detected proAMH, with AMHN,C cross-detection conservatively estimated at 6.0% ± 2.5% (mean ± S.D.). The intra-assay and inter-assay variability were estimated at 8.0%CV and 13.0%CV respectively. The levels of proAMH and total AMH were assessed in 5 boys and 5 men and the proportion of proAMH was found to be significantly higher in boys (p = 0.005). This study will facilitate further investigation of the role of proteolytic cleavage in AMH signalling.