Previous studies have shown that desialylation of human chorionic gonadotropin (hCG) results in a sharp enhancement of its affinity for thyroid thyroid-stimulating hormone (TSH) receptors, transforming it from a weak to a potent antagonist of adenylate cyclase activity in vitro. Because most of the information on the structure-function relation of hCG as a thyroid stimulator has been derived from in vitro experiments, the present studies were undertaken to assess the role of its sialic acid residues in the expression of its thyrotropic activity in vivo. hCG and its various desialylated forms, viz., intact-alpha-asialo-beta, asialo-alpha-intact-beta, and asialo-hCG (ashCG), were initially characterized in terms of their immunoreactivities and receptor-binding abilities as assessed in the rat testis assay. In neither assay did hCG or its variants exhibit a major discordance in activity. In the mouse bioassay, intact hCG (150 micrograms) proved to be a thyroid stimulator of considerable potency, exceeding the response induced by 0.2 mIU bovine TSH (bTSH), as measured by 125I release into the blood after 2- and 8-h intervals. Remarkably, both asialo-alpha-intact-beta and ashCG significantly stimulated the mouse thyroid in this assay, though to a lesser degree than hCG itself. However, in the same assay intact-alpha-asialo-beta was inactive. Studies of the survival of hCG and its variants in the circulation of the mouse, as assessed by radioimmunoassay (RIA) in multiple serum samples drawn over 30 min, showed hCG to have a long half-life, whereas ashCG was cleared very rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)
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
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