A New Method for Assessing the Thyrotropin Binding Inhibitory Activity in the Immunoglobulins and Whole Serum of Patients with Graves' Disease*

Abstract

Immunoglobulin G (IgG) fractions prepared from the serum of patients with Graves' disease (Graves'-IgG) are generally capable of inhibiting the binding of 125 I-labeled bovine TSH ([ 125 I]bTSH) to crude preparations of human thyroid membranes [TSH binding inhibitory (TBI) activity]. In current TBI assays, membranes, [ 125 I]bTSH, and IgG are incubated together, and the extent of inhibition of [ 125 I]bTSH binding produced by Graves'-IgG is compared with that produced by specimens of normal IgG. Such direct TBI assays, as we term them, have only moderate sensitivity, positive results being reported in approximately 50-75% of the actively thyrotoxic patients with Graves' disease. This appears to be owing to the wide ranging and often substantial TBI activity displayed by preparations of normal IgG.Reasoning that the TBI activity of normal IgG might be more readily dissociable from the thyroid membranes than the IgG specific for Graves' disease would be, we modified the TBI assay by first incubating the membranes with or without IgG, washing them thoroughly with buffer, and then incubating them with [ 125 I]bTSH. We term this a residual TBI assay, since it tests the extent of TBI activity that remains associated with the membranes despite the washing procedure. This procedure greatly reduced or eliminated the TBI activity of normal IgG and yielded a 94% frequency of positive responses in studies of 50 specimens of Graves'-IgG. In 31 specimens so tested, values of the residual TBI assay correlated significantly with their ability to increase the cAMP concentration in human thyroid slices.Ensuing experiments were conducted to test the feasibility of applying the principle of the residual TBI assay to the assay of whole serum, rather than IgG. A modification of the washing procedure used in residual TBI assays of IgG was shown to greatly decrease the almost complete inhibitory activity of serum seen in direct TBI assay. In residual TBI assays of 35 specimens of whole serum from patients with Graves' disease, a 77% frequency of positive responses was observed. In 27 samples so studied, a significant correlation was observed between the TBI activity of Graves'-IgG and that of the sera from which they were prepared. Application of the residual assay principle affords promise of greatly simplifying and enhancing the sensitivity of TBI assays. (J Clin Endocrinol Metab 54: 552, 1982) I T IS generally agreed that the thyroid hyperfunction in Graves' disease results from the action on the thyroid of abnormal immunoglobulins (Igs) of the IgG class that interact with the thyroid plasma membrane. As judged from in vitro studies, this interaction variously results in an activation of adenylate cyclase and an inhibition of the binding of TSH to its specific receptor in the plasma membrane, possibly owing to binding of these IgG to the receptor itself. These responses form the basis for the two types of assay most commonly employed at present to detect these disease-specific IgGs. One

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.

“…Graves' disease is associated with the production and circulation of heterogenous antibodies, some of which are directed against thyroid-stimulating hormone (TSH)' receptors located in thyroid gland membranes (1)(2)(3)(4)(5)(6)(7)(8)(9). Recent investigations have proposed that a subset of these antibodies may bind to certain epitopes on TSH receptors and cause unabated stimulation, thereby resulting in thyroid hyperplasia and hypersecretion, the end result of which is detected as an elevated serum 3,5,3',5'-tetraiodothyronine (T4) and 3,5,3'-triiodothyronine (T3) and clinical thyrotoxicosis (1)(2)(3)(4)(5)(6)(7)(8)(9).…”

“…Recent investigations have proposed that a subset of these antibodies may bind to certain epitopes on TSH receptors and cause unabated stimulation, thereby resulting in thyroid hyperplasia and hypersecretion, the end result of which is detected as an elevated serum 3,5,3',5'-tetraiodothyronine (T4) and 3,5,3'-triiodothyronine (T3) and clinical thyrotoxicosis (1)(2)(3)(4)(5)(6)(7)(8)(9).…”

“…Currently, most assays developed to detect these heterogenous antibodies measure either the inhibition of '251-TSH binding or the stimulation of thyroidal adenylate cyclase (1)(2)(3)(4)(5)(6)(7)(8)(9). Studies measuring inhibition of 1251-TSH binding to thyroid gland membranes have greatly advanced our understanding of the pathophysiology of Graves' disease (1-3, 5, 6, 8, 9), but suffer, in general, from several major disadvantages: (a) They require fractionation of serum with extraction of the immunoglobulins; (b) '25I-TSH binding inhibition represents an indirect method of assessing receptor-antibody interactions and it is not certain that direct binding assays would result in comparable findings; (c) '251-labeled bovine TSH is routinely used rather than a system that directly assesses the relationship between human TSH and its receptor; and (d) these assays, which frequently use protein A-Sepharose columns for serum fractionation, would detect immunoglobulins of only some IgG subclasses and would probably not determine whether other classes (e.g., IgM) play an important role in the receptor-immunoglobulin interactions in Graves' disease.…”

“…IgG fractions were prepared by separation of whole serum on columns of Protein-A-sepharose Cl-4B by methods previously reported (9,12,13). IgG fractions prepared in this manner were subdivided into aliquots and kept frozen at -70°C until used.…”

“…The thyroid glands were frozen at -70°C within 30 min of their removal. On a separate day, the glands were thawed and membranes were prepared as described previously (9,(12)(13)(14). In brief, minced thyroid gland in icecold 1 (14).…”