Kakinuma A, Chazenbalk G D, Jaume J C, Rapoport B, McLachlan S M
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
J Clin Endocrinol Metab. 1997 Jul;82(7):2129-34. doi: 10.1210/jcem.82.7.4092.
Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100-200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves' disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.
在人类促甲状腺激素受体（TSHR）分子克隆七年后，猪TSHR仍普遍用于TSHR自身抗体的促甲状腺激素结合抑制（TBI）试验。我们在两种类型的TBI试验中比较了猪TSHR和重组人TSHR：一种使用在其表面表达重组人TSHR的完整中国仓鼠卵巢细胞，另一种使用用去污剂从这些细胞中提取的可溶性受体。在完整细胞TBI试验中，表达大量TSHR的单层细胞比表达少量受体的细胞效果差。这些发现与血清中TSHR自身抗体的极低浓度一致。[125I]人促甲状腺激素与完整细胞的结合比[125I]牛促甲状腺激素低约5倍。然而，对于大多数血清，两种配体的TBI值相似。然而，少数血清对人促甲状腺激素结合的抑制作用比对牛促甲状腺激素结合的抑制作用更大。在可溶性人TSHR TBI试验中，与完整细胞TBI试验相反，表达大量TSHR的细胞是用去污剂提取可溶性人TSHR的极好来源，但前提是细胞仍在培养皿上时进行提取，而不是刮下后提取。当在商业TBI试剂盒中替代可溶性猪TSHR时，一个直径10厘米的细胞培养皿提供的TSHR可用于100 - 200次重复测定。用可溶性人和猪TSHR检测时，30名疑似格雷夫斯病患者血清中的TBI值密切相关（r = 0.954；P < 0.001）。然而，2份对猪TSHR呈阴性的血清对人TSHR呈阳性。无论TBI使用人TSHR还是猪TSHR，这些血清中的TBI和甲状腺刺激活性相关性都较弱。这些发现为使用重组人TSHR进行实际的TBI试验开辟了道路。
