Immunoprecipitation of Radiolabeled Human Thyroid Cell Proteins by Serum from Patients with Autoimmune Thyroid Disease*

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Abstract
Characterization of the antigen to thyroid-stimulating Ig (TSI), presumably the TSH receptor, remains elusive. Therefore, immunoprecipitation of the TSI antigen was attempted using cultured human thyroid cells radiolabeled to high specific activity by [35S]methionine (all proteins labeled) or by 125I and lactoperoxidase (primarily cell surface proteins labeled). Both techniques produced similar results. Crude membrane preparations from the labeled cells were solubilized in buffer containing 1% Triton, and then incubated with TSI-containing or control serum. Ig was precipitated with goat antihuman Ig and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 30 proteins were immunoprecipitated by both normal and control sera. However, only 1 protein (MW, .apprx. 320,000) was preferentially selected by TSI-containing serum (6 of 6 tested). This protein was also immunoprecipitated by the majority of sera from patients with Hashimoto''s thyroiditis. The 320,000 MW protein constituted only a minute amount of total radiolabeled membrane protein, because it was not apparent on electrophoresis of the membrane extract before immunoprecipitation. It corresponded (in migration) to a human thyroglobulin (Tg) standard. Tg also inhibited immunoprecipitation of the 320,000 MW band. The anti-Tg antibody titer correlated significantly with the degree of immunoprecipitation of the 320,000 MW band. No specific immunoprecipitation of any protein was observed when radiolabeled guinea pig membrane proteins were used. These data provide insight into the difficulties involved in the identification of the TSH receptor with serum containing TSI.