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 ¹²⁵I-labeled bovine TSH ([¹²⁵I]bTSH) to crude preparations of human thyroid membranes [TSH binding inhibitory (TBI) activity]. In current TBI assays, membranes, [¹²⁵I]bTSH, and IgG are incubated together, and the extent of inhibition of [125I]bTSH binding produced byGraves'-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 [¹²⁵I]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 principleof 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.