Whitfield G K, Miller W L
Endocrinology. 1984 Jul;115(1):154-9. doi: 10.1210/endo-115-1-154.
An assay was developed to detect tritium-labeled ovine FSH beta-subunit [( 3H]oFSH beta) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSH beta (RCM-oFSH beta) in a two-cycle immunoextraction procedure. A discrete species with an apparent mol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM-[3H]oFSH beta by its comigration with highly purified RCM-oFSH beta, its reduction in culture media after cultures were treated with 17 beta-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSH beta. The assay was used in a pulse-chase study to determine that [3H]oFSH beta is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17 beta-estradiol did not change this timing of secretion.
开发了一种检测从原代绵羊垂体培养物中分泌的氚标记的绵羊促卵泡激素β亚基[³H]oFSHβ的分析方法。该程序在两循环免疫提取过程中使用了针对还原和氨甲酰甲基化的oFSHβ（RCM-oFSHβ）产生的亲和富集抗体。在来自培养基的免疫提取物的十二烷基硫酸钠电泳图谱中检测到一种表观分子量为21,000的离散条带。通过与高度纯化的RCM-oFSHβ共迁移、在用17β-雌二醇处理培养物后其在培养基中的减少（这通常会降低放射免疫可检测的oFSH）以及被过量未标记的RCM-oFSHβ从提取抗体中置换出来，该条带被鉴定为RCM-[³H]oFSHβ。该分析方法用于脉冲追踪研究，以确定[³H]oFSHβ在其合成后1-2小时内被分泌。用17β-雌二醇预先处理培养物并没有改变这种分泌时间。
