1. Enzyme-linked immunosorbent assay measurements of antimüllerian hormone (AMH) in human blood are a composite of the uncleaved and bioactive cleaved forms of AMH 
Michael W. Pankhurst, Ph.D., Yih Harng Chong, M.B.Ch.B., Ian S. McLennan, Ph.D. 
Fertility and Sterility 
Volume 101, Issue 3, Pages e1 (March 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
The recombinant proAMH (lane 2, both blots) and AMHN,C (lane 2, both blots) preparations were run on Western blots to demonstrate the relative quantities of proAMH, AMHN, and AMHC present. (A) The N-terminal antibody detects proAMH and AMHN bands as indicated by the labeled arrows. (B) The C-terminal antibody detects the proAMH and the AMHC bands (labeled arrows). Trace levels of AMH255–560 (labeled arrow) arise from alternative cleavage variant of AMH (14) that is not found in detectable quantities in human blood (16). Lane 1 in both blots is the molecular weight marker. (C) The activity of the recombinant AMH preparation was tested in a bioassay. Various concentrations of recombinant AMHN,C were applied to P19 cells expressing an AMH reporter gene. The firefly luciferase reading was standardized to a transfection-control Renilla luciferase reading for each well. Concentrations shown are averaged from duplicate treatments.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
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3. Figure 2
The different forms of AMH captured by the Gen II ELISA plates were recovered and visualized by Western blot. The Western blots were probed with antibodies to both the N-terminal and C-terminal domains of AMH. The plates were incubated with the proAMH preparation which contained traces of AMHN,C (lane 1), an AMHC-only preparation (lane 2), an AMHN-only preparation (lane 3), and human serum (lane 4). Lane 4 is shown from a scan with a higher gain to detect the lower concentration of AMH in this sample. Molecular weight marker migration is shown on the far left column.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Supplemental Figure 1
Recombinant human proAMH is not readily cleaved to AMHN,C in human serum. The proAMH (370 pM) was incubated for 24 hours at 37°C in control buffer (1% BSA in phosphate-buffered saline), heat inactivated human serum (56°C, 1 hour to inactivate endogenous proteases), or untreated human serum. ProAMH and any AMHN,C was immunoprecipitated for 1 hour with 1 μg of goat anti-AMH-N-terminal domain antibody followed by Western blot with the same antibody to detect both proAMH and AMHN, as described elsewhere (13). The immunoprecipitating antibody band is indicated by the blue arrowhead. The immunoprecipitation recovered similar amounts of proAMH from the control, the heat inactivated serum, and untreated serum sample. The amount of AMHN was not greater in the serum sample, which indicated that no appreciable proAMH cleavage to AMHN,C had occurred. This indicates that proAMH is stable for at least 24 hours. The increases in AMH values from the AMH Gen II ELISA after samples are prediluted are unlikely to be due to cleavage of proAMH.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions