The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies.
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.
Kakinuma
A
A
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
Chazenbalk
G D
GD
Jaume
J C
JC
Rapoport
B
B
McLachlan
S M
SM
D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001667:Binding, Competitive; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D006801:Humans; D008722:Methods; D011989:Receptors, Thyrotropin / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013972:Thyrotropin / Q000378:metabolism*; D014162:Transfection
D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin