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

Abstract

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) was developed. gamma 2-MSH (1-12) was biotinylated, trapped onto an anti-gamma 2-MSH (1-12) IgG-coated polystyrene bead, eluted at pH 1 after washing to eliminate other biotinylated substances, and measured using two streptavidin-coated polystyrene beads and affinity-purified anti-gamma 2-MSH (1-12) Fab'-peroxidase conjugate. The detection limit of gamma 2-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 alpha-MSH, beta-MSH, and gamma 1-MSH. By this immunoassay, the concentration and molecular size of immunoreactive gamma 2-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive gamma 2-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 gamma 2-MSH (1-12) as standard. Immunoreactive gamma 2-MSH detected by the noncompetitive enzyme immunoassay after removal of high molecular weight substances was not homogeneous and smaller than gamma 2-MSH (1-12), and its concentration was approximately 1 pmol/L. The exact nature of these immunoreactive gamma 2-MSHs remains to be elucidated. gamma 2-MSH (1-12) added to plasma was degraded rapidly, and the concentration of gamma 2-MSH (1-12) was very low, if any, in plasma of healthy subjects.