A sensitive method for assaying thyroid stimulating immunoglobulins of Graves' disease: use of the guanyl nucleotide-amplified thyroid adenylate cyclase assay

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Abstract
A sensitive method for evaluating adenylate cyclase stimulation by thyroid-stimulating antibodies (TSAb) was developed and validated, based on the measurement of thyroid membrane adenylate cyclase activity in the presence of a non-hydrolyzable GTP analog, guanyl-5''-yl imidodiphosphate (Gpp(NH)p). The addition of Gpp(NH)p (10-5 M) produced a 10-fold increase of the sensitivity of the system for both TSH and TSAb. IgG preparations from sera of 30 patients with Graves'' disease were tested for the adenylate cyclase stimulation either in the presence or in the absence of Gpp(NH)p: a significant stimulation was observed in 27/30 patients when the GTP analog was added to the system, while only 20/30 patients were positive in the absence of the nucleotide. The advantage of Gpp(NH)p addition was also evident in a large series which included 57 patients with Graves'' disease, 15 with Hashimoto''s thyroiditis or primary myxedema and 22 normal subjects. In fact, 88% of patients with Graves'' disease resulted positive, while no significant stimulation was elicited by Hashimoto''s thyroiditis, primary myxedema and by normal immunoglobulins. The sensitivity achieved in the system which employs thyroid plasma membranes was similar to that obtained by other investigators with the use of thyroid slices or thyroid cells in primary culture. Furthermore, methods based on thyroid plasma membranes are supposed to have a better reproducibility, since the same tissue preparation, if appropriately stored, may be used in several different tests.