Functionality of thyroid-stimulating antibodies assessed by cryopreserved human thyroid cell bioassay

Immunoglobulin preparations from patients with autoimmune thyroid disease may inhibit the binding of ¹²⁵I-labeled bovine TSH to solubilized TSH receptors without exhibiting concurrent thyroid stimulation. Such data have emphasized the continuing need for assay of biological function in addition to binding inhibition. We have employed a convenient technique using normal human thyroid cells cultured in mono-layers for 7 days and then cryopreserved in liquid N₂. Thyroid stimulation was assessed by measuring intracellular cAMP accumulation in a modified hypotonic Hank’s Balanced Salt Solution using approximately 30,000 cells/well. Forty-five consecutive unselected immunoglobulin samples from hyperthyroid Graves’ patients were assayed at 10 mg/ml protein for 2 h. Thirty-two (71%) of these untreated samples and 3 of 13 (23%) samples from treated euthyroid Graves’ patients caused significantly greater stimulation than normal Ig prepared and assayed simultaneously. Forty-four percent of the positive samples were of low titer, causing less than a 200% increase in basal cAMP levels. One high titer sample (12,000% increase) had even greater bioactivity when serially diluted, thus suggesting the presence of an inhibitor, a phenomenon not observed in 20 lower titer and negative preparations. These data indicate the convenience and usefulness of a cryopreserved thyroid cell bioassay and emphasize that only low titers of thyroid-stimulating antibody occur in the peripheral circulation of many Graves’ patients.