Yagura T, Walfish P G
Biochem J. 1982 Dec 15;208(3):641-9. doi: 10.1042/bj2080641.
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-[(125)I]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.
采用一种新的未提取样品放射免疫分析法，利用8-苯胺基萘-1-磺酸抑制肝核提取物中L-[(125)I]三碘甲状腺原氨酸与核L-三碘甲状腺原氨酸受体的结合，测定肝核提取物中的内源性L-三碘甲状腺原氨酸含量。对于该方法，最低检测限为3.125 pg/管，添加的L-三碘甲状腺原氨酸回收率为90%-120%，批间变异系数为10%。正常甲状腺大鼠肝核提取物中内源性L-三碘甲状腺原氨酸含量为10-40 pg/0.2 ml，而甲状腺切除大鼠的含量低于3.125 pg/0.2 ml。将该新方法得到的结果与葡聚糖凝胶G-25柱提取样品放射免疫分析法进行比较，结果吻合良好。利用内源性L-三碘甲状腺原氨酸含量的值对结合测定混合物中的L-三碘甲状腺原氨酸浓度进行校正，以便通过Scatchard分析准确确定核L-三碘甲状腺原氨酸受体的结合特性。通过使用甲状腺切除大鼠的核提取物来证明内源性L-三碘甲状腺原氨酸校正的有效性，该提取物在测定核L-三碘甲状腺原氨酸受体结合特性之前，先与少量已知量的L-三碘甲状腺原氨酸预孵育。当对预孵育剂量进行Scatchard作图校正时，得到的结果与真实值相似，但未校正时结果会被错误地低估。结论是，已证明在Scatchard分析计算核L-三碘甲状腺原氨酸受体结合特性时使用内源性L-三碘甲状腺原氨酸校正的必要性和有效性，对于亲和常数而言，这种校正比对最大结合容量更为重要。
