Functionality of Thyroid-Stimulating Antibodies Assessed by Cryopreserved Human Thyroid Cell Bioassay*

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Abstract
Ig 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. A convenient technique using normal human thyroid cells cultured in monolayers for 7 days and then cryopreserved in liquid N2 was employed. Thyroid stimulation was assessed by measuring intracellular cAMP accumulation in a modified hypotonic Hank''s Balanced Salt Solution using .apprx. 30,000 cells/well. Forty-five consecutive unselected Ig 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.

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