To obviate several problems inherent in indirect thyroid-stimulating hormone (TSH) receptor antibody assays, we developed an enzyme-linked immunosorbent assay (ELISA) that measures antibodies binding to guinea pig fat cell membrane, which contain high concentrations of TSH receptors. Solubilized guinea pig fat cell membranes were adsorbed to plastic microtiter plates and served as the solid-phase antigen. Test sera and affinity-purified alkaline phosphatase-conjugated anti-human IgG were co-incubated with membranes, after which p-nitrophenyl phosphate was added. Results were read when a positive control reached a standard color change (OD(405nm)). Specificity of this assay was demonstrated by the inability of albumin, insulin, TSH subunits, propranolol, or dexamethasone to block binding. 30 normal subjects had a mean OD value of 0.080 ± 0.050 (SD). 23 of 25 untreated Graves' patients had OD values at least 2 SD above the normal mean (Grave's mean ± SD; 0.46 ± 0.33, P < 0.001) and in each case 10-6 M TSH inhibited the binding by at least 60%, suggesting that the immunoglobulins were directed at the TSH receptor. Seven of 25 serum samples from patients with Hashimoto's disease, seven of 23 serum samples from patients with transient hyperthyroidism (subacute thyroiditis or painless thyrotoxic thyroiditis), and two of 10 samples from patients with thyroid carcinoma had significant elevations in the titers of membrane-directed immunoglobulins. Graves' patients who were treated with ablative therapy at least 6 mo earlier and who were euthyroid when restudied continued to have abnormally elevated membrane-directed immunoglobulins in six of eight samples studied. Further studies involved the substitution of affinity-purified alkaline phosphatase anti-IgM antisera for the anti-IgG antisera routinely used. Seven of 12 serum samples from patients with Graves' disease had significant elevations in binding which in every instance was inhibited by >60% by 10-6 M TSH. In sum, the present results indicate that (a) we have developed a sensitive, specific, reproducible, convenient ELISA for the measurement both of the total amount of circulating membrane-directed antibodies and of TSH-displaceable membrane-directed immunoglobulins. (b) This ELISA detected significant elevations in TSH-displaceable guinea pig membrane binding in 23 of 25 untreated Graves' patients as well as in ~30% of patients with Hashimoto's thyroiditis and subacute thyroiditis. (c) Elevated membrane directed antibodies may continue to be present many months or years after restoration of the euthyroid state. (d) Circulating membrane binding IgM immunoglobulins have been detected in patients with Graves' disease. Further studies using this ELISA should prove useful in a variety of investigative and clinical studies.
ASJC Scopus subject areas