Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.
Precursor forms of the alpha-subunit of inhibin are abundant in human follicular fluid and possibly plasma, although their function is uncertain. We now describe the development of a new enzyme-linked immunosorbent assay to measure inhibin forms containing both the pro and alpha C regions of the alpha-subunit. The assay has a detection limit for purified human pro-alpha C of 0.5 pg/mL and less than 0.02% cross-reaction with recombinant forms of inhibin, activin, and follistatin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of follicular fluid extracts demonstrated that the assay is likely to detect pro-containing precursor forms of both the free alpha-subunit and intact dimeric inhibin. The serum concentration was measured in normal men (446 +/- 28 pg/mL), postmenopausal women (45.8 +/- 3.8 pg/mL), and women treated with FSH before in vitro fertilization (1827 pg/mL). Pooled human follicular fluid contained 488 ng/mL. The mean serum concentration in the female menstrual cycle rose from 150.6 +/- 26.1 pg/mL in the early follicular phase to 692.2 +/- 113 pg/mL in the midluteal phase. This assay offers a useful tool for investigation of the role of inhibin-related proteins in human reproduction. There may be particular clinical value under circumstances in which other assays for inhibin forms have insufficient sensitivity.
Groome
N P
NP
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, United Kingdom.
Illingworth
P J
PJ
O'Brien
M
M
Priddle
J
J
Weaver
K
K
McNeilly
A S
AS
D028341:Activins; D000595:Amino Acid Sequence; D000906:Antibodies; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D038681:Follistatin; D006023:Glycoproteins; D006801:Humans; D015151:Immunoblotting; D007265:Inhibins / Q000097:blood* / Q000302:isolation & purification; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D017698:Postmenopause; D011498:Protein Precursors / Q000097:blood* / Q000302:isolation & purification; D011994:Recombinant Proteins; D012016:Reference Values; D012680:Sensitivity and Specificity; D012727:Sex Characteristics
D000906:Antibodies; D038681:Follistatin; D006023:Glycoproteins; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C095971:inhibin-alpha subunit precursor; D028341:Activins; D007265:Inhibins