Noncompetitive enzyme immunoassay (hetero‐two‐site enzyme immunoassay) for γ‐melanocyte‐stimulating hormone (γ‐msh) and measurement of immunoreactive γ‐msh in plasma of healthy subjects

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for γ-melanocyte-stimulating hormone (γ-MSH) was developed. γ-MSH (1–12) was biotiny-lated, trapped onto an anti-γ-MSH (1–12) IgG-coated polystyrene bead, eluted at pH 1 after washing to eliminate other biotiny-lated substances, and measured using two streptavidin-coated polystyrene beads and affinity-purified anti-γ-MSH (1–12) Fab'-per-oxidase conjugate. The detection limit of γ-MSH (1–12) was 10–30 amol (16–48 fg)/assay and 130–400 fmol (210–630 pg)/L of plasma. There was little or only slight cross reaction with α-MSH, b-MSH, and -MSH. By this immunoassay, the concentration and molecular size of immunoreactive γ-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive γ-MSH measured by competitive enzyme immunoassay was a mixture of substances with high molecular weights (100–500 kDa), and its concentration was calculated to be 50–60 pmol/L using γ-MSH (1–12) as standard. Immunoreactive γ-MSH detected by the noncompetitive enzyme immunoassay after removal of high molecular weight substances was not homogeneous and smaller than γ-MSH (1–12), and its concentration was ∼ 1 pmol/L. The exact nature of these immunoreactive γ-MSHs remains to be elucidated. γ-MSH (1–12) added to plasma was degraded rapidly, and the concentration of γ-MSH (1–12) was very low, if any, in plasma of healthy subjects.