Partial characterization and clinical correlation of circulating human immunoglobulins directed against thyrotrophin binding sites in guinea pig fat cell membranes. Development of a direct enzyme immunoassay

The Journal of Clinical Investigation
J R BakerK D Burman

Abstract

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 (OD405nm). 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 less than 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 patient...Continue Reading

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