Faivre-Bauman A, Gourdji D, Tixier-Vidal A
Ann Endocrinol (Paris). 1977 Jul-Aug;38(4):265-71.
The high affinity receptor for TRH of a rat pituitary GH3 strain in culture (Km: 4 X 10(-9) M) was found suitable to design a radio-receptor assay for TRH. Attached GH3 cells were incubated for 30 min at 37 degrees C in presence of a tracer amount of 3H-TRH (200 pg/ml, 0.54 nM) and increasing quantities of cold synthetic TRH. After extensive washings, the radioactivity associated with the cells was counted, and the log-logit transformation was applied to the results expressed as % binding (100%: binding in absence of unlabelled TRH). The standard curve is linear between 200 pg/ml and 5 ng/ml TRH. Hypothalamic or thyroid hormones were tested for their competitive effect with 3H-TRH towards the GH3 cells receptor. None of them interfered. TRH analogs were also assayed for their competition with TRH towards the receptor. Only compounds methylated on Prolineamide or Histidine could compete with TRH, but these derivatives have never been shown to occur in vivo. Because of this high specificity, we applied this radioreceptor assay to biological samples, once extracted by acidic methanol and purified by CMC column (following a technics designed by K Bauer, Berlin).
培养的大鼠垂体GH3细胞系中促甲状腺激素释放激素（TRH）的高亲和力受体（Km：4×10⁻⁹ M）被发现适合用于设计TRH的放射受体测定法。将贴壁的GH3细胞在37℃下与示踪量的³H-TRH（200 pg/ml，0.54 nM）和不断增加量的冷合成TRH一起孵育30分钟。经过大量洗涤后，对与细胞相关的放射性进行计数，并对以结合百分比（100%：无未标记TRH时的结合）表示的结果进行对数-对数转换。TRH的标准曲线在200 pg/ml至5 ng/ml之间呈线性。测试了下丘脑或甲状腺激素与³H-TRH对GH3细胞受体的竞争作用。它们均无干扰。还测定了TRH类似物与TRH对受体的竞争作用。只有脯氨酰胺或组氨酸甲基化的化合物能与TRH竞争，但这些衍生物从未在体内被证明存在。由于这种高特异性，我们将这种放射受体测定法应用于生物样品，生物样品先用酸性甲醇提取，然后通过羧甲基纤维素柱纯化（按照柏林的K·鲍尔设计的技术）。
