Noncompetitive enzyme immunoassay (hetero-two-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 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.
Yogi
Y
Y
First Department of Surgery, Miyazaki Medical College, Japan.
Hashida
S
S
Ekman
R
R
Setoguchi
T
T
Ishikawa
E
E
D000328:Adult; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001710:Biotin; D005069:Evaluation Studies as Topic; D006801:Humans; D007120:Immunochemistry; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D008297:Male; D009074:Melanocyte-Stimulating Hormones / Q000097:blood* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012016:Reference Values; D012680:Sensitivity and Specificity
D001710:Biotin; D009074:Melanocyte-Stimulating Hormones