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A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

机译:一种新的未提取样品的放射免疫分析方法,用于测定肝内源性核内的l-三碘甲状腺素含量。用于确定核受体结合特性的有效性

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摘要

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[125I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.
机译:通过一种新的未萃取样品放射免疫分析方法,使用8-苯胺基萘-1-磺酸抑制L- [125I] tri-碘代甲状腺素与核L-tri-三核素的结合,测量肝核提取物中的内源性L-三碘甲状腺素含量。提取物中的碘甲状腺素受体。对于该方法,灵敏度下限为3.125 pg /管,添加的l-三碘甲状腺素的回收率为90–120%,测定间变异系数为10%。正常甲状腺大鼠的内源性l-三碘甲状腺素含量为10–40 pg / 0.2 ml肝核提取物,而接受甲状腺切除的大鼠的内源性l-三碘甲状腺氨酸的含量少于3.125 pg / 0.2 ml。将该新方法获得的结果与Sephadex G-25柱提取样品放射免疫分析方法进行比较,并显示出良好的一致性。利用内源I-三碘甲状腺素含量的值校正结合测定混合物中的I-三碘甲状腺素浓度,以便通过Scatchard分析准确确定核I-三碘甲状腺素受体的结合特性。通过使用来自甲状腺切除的大鼠的核提取物证明内源性l-三碘甲状腺素校正的有效性,该大鼠的核提取物在确定核l-三碘甲状腺素受体结合特征之前与少量已知的l-三碘甲状腺素进行了预孵育。当对Scatchard图进行预温育剂量校正时,获得的结果与真实值相似,但是如果不进行校正,则错误地降低了结果。结论是,已经证明在Scatchard分析计算核I-三碘代甲状腺素受体结合特性中使用内源性I-三碘代甲状腺素校正的必要性和有效性,对于亲和力常数比最大结合能力尤为重要。

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