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, b̃‐MSH, and   γ   1‐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.