NONCOMPETITIVE ENZYME-IMMUNOASSAY (HETERO-2-SITE ENZYME-IMMUNOASSAY) FOR GAMMA-2-MELANOCYTE-STIMULATING HORMONE (GAMMA-2-MSH) AND MEASUREMENT OF IMMUNOREACTIVE GAMMA-2-MSH IN PLASMA OF HEALTHY-SUBJECTS

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassa y) for p-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 str eptavidin-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 (210-63 0 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 conc entration 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 p-MSH measure d by competitive enzyme immunoassay was a mixture of substances with h igh molecular weights (100-500 kDa), and its concentration was calcula ted to be 50-60 pmol/L using (gamma 2)-MSH (1-12) as standard. Immunor eactive (gamma 2)-MSH detected by the noncompetitive enzyme immunoassa y after removal of high molecular weight substances was not homogeneou s and smaller than (gamma 2)-MSH (1-12), and its concentration was sim ilar to 1 pmol/L. The exact nature of these immunoreactive (gamma 2)-M SHs 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 v ery low, if any, in plasma of healthy subjects. (C) 1995 Wiley-Liss, I nc.