Preparation of125I-Labeled Receptor-Purified Graves' Immunoglobulins: Properties of Their Binding to Human Thyroid Membranes*

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Abstract
We have recently reported a method for the receptor purification of Graves' disease-specific immunoglobulin G (IgG) using plasma membranes from guinea pig adipocytes as a source of TSH receptor. The preparations of receptor-purified Graves'-IgG (RPG-IgG) thereby obtained were highly enriched (40- to 150-fold) in TSH binding inhibitory activity and were comparably enriched in human thyroid adenylate cyclase-stimulating activity. Unlike the less active preparations obtained by others who have attempted receptor purification but have used human thyroid membranes, our preparations were devoid of the antimicrosomal antibodies and, presumably, the antithyroglobulin antibodies common to the IgG of patients with Graves' and Hashimoto's diseases. We have now succeeded in preparing 125Ilabeled RPG-IgG (RPG-IgG*) by a lactoperoxidase method that permits retention of the original TSH binding inhibitory activity. Such preparations have been employed to study both the direct binding of Graves'-specific IgG to human thyroid membranes and several of the properties of this interaction. The binding of RPG-IgG* to human thyroid membranes exceeded that of 125I-labeled receptor-purified normal IgG and was saturable, since it could be inhibited almost entirely by sufficient concentrations of unlabeled Graves'-IgG. The binding of RPG-IgG* prepared in the standard manner (i.e. with adipocyte membranes) was inhibited by Graves'-IgG, but not by Hashimoto's IgG, when tested in either human thyroid membranes or guinea pig adipocyte membranes. RPG-IgG* prepared with the use of human thyroid membranes exhibited the same differential inhibition of binding when tested in adipocyte membranes, but was inhibited by both Graves'- and Hashimoto's-IgG when tested in human thyroid membranes. We interpret these findings to indicate that, in general, human thyroid membranes contain antigens that are complementary to antibodies present in the serum in both Graves' and Hashimoto's diseases, while adipocyte membranes contain antigens complementary to antibodies found in the serum in Graves' disease, but not in Hashimoto's disease. The binding of RPG-IgG* prepared with adipocyte membranes and the binding of 125I-labeled bovine TSH (bTSH*) to human thyroid membranes had several features in common. Like the binding of bTSH*, that of RPG-IgG* was temperature dependent, had a pH optimum of about 6.0, and was progressively inhibited by increasing concentrations of NaCl. In membranes from a variety of tissues, saturable binding of RPG-IgG* was greatest in those with the greatest binding of bTSH*. Moreover, the ability of individual specimens of Graves' IgG to inhibit the binding of RPG-IgG* to human thyroid membranes was closely correlated with their ability to inhibit the binding of bTSH*. Finally, the studies appeared to provide the first demonstration of the ability of unlabeled bovine and human TSH to inhibit the binding of RPG-IgG*, though inhibition was only partial and relatively high concentrations were required. Even higher concentrations of other peptide hormones were without effect, however. The studies have provided, therefore, both the first direct demonstration of saturable binding of Graves'-specific IgG to human thyroid membranes and strong evidence that such binding occurs at the TSH receptor, though perhaps not exclusively so. The preparations of RPG-IgG* that we have described could possibly be used as a reagent in radioreceptor assays for Graves'-specific IgG.

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