Yogi Y, Hashida S, Ekman R, Setoguchi T, Ishikawa E
First Department of Surgery, Miyazaki Medical College, Japan.
J Clin Lab Anal. 1995;9(6):397-406. doi: 10.1002/jcla.1860090611.
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.
已开发出一种用于γ2-促黑素细胞激素（γ2-MSH）的非竞争性酶免疫测定法（异双位点酶免疫测定法）。γ2-MSH（1-12）被生物素化，捕获到抗γ2-MSH（1-12）IgG包被的聚苯乙烯珠上，洗涤后在pH 1下洗脱以去除其他生物素化物质，并使用两个链霉亲和素包被的聚苯乙烯珠和亲和纯化的抗γ2-MSH（1-12）Fab'-过氧化物酶缀合物进行测量。γ2-MSH（1-12）的检测限为每次测定10-30 amol（16-48 fg）和血浆中130-400 fmol（210-630 pg）/L。与α-MSH、β-MSH和γ1-MSH几乎没有或只有轻微的交叉反应。通过这种免疫测定法，检测了健康受试者血浆中免疫反应性γ2-MSH的浓度和分子大小，并将结果与竞争性酶免疫测定法的结果进行了比较。通过竞争性酶免疫测定法测量的免疫反应性γ2-MSH是高分子量（100-500 kDa）物质的混合物，以γ2-MSH（1-12）为标准计算其浓度为50-60 pmol/L。去除高分子量物质后通过非竞争性酶免疫测定法检测到的免疫反应性γ2-MSH不均匀且小于γ2-MSH（1-12），其浓度约为1 pmol/L。这些免疫反应性γ2-MSH的确切性质仍有待阐明。添加到血浆中的γ2-MSH（1-12）迅速降解，健康受试者血浆中γ2-MSH（1-12）的浓度非常低（如果有的话）。
