Development and Evaluation of a Method for the Partial Purification of Immunoglobulins Specific for Graves’ Disease*

Abstract

Sera of patients with Graves' disease contain immunoglobulins (IgG) that bind to thyroid plasma membranes, inhibiting thereby the binding of [¹²⁵I]bovine TSH to its receptors [TSH-binding inhibitory IgG (TBII)], activate adenylate cyclase, or do both. In this report, we describe a new method for the extensive purification of IgG specific to the serum of patients with Graves' disease, using receptor purification as the isolation technique and the TBII assay as an index of activity. Human thyroid tissue was considered an undesirable source of receptor for two reasons. First, the IgG of sera of patients with Graves' disease almost always contain antimicrosomal and/or antithyroglobulin antibodies found in patients with other thyroid diseases, especially Hashimoto's thyroiditis. Second, many preparations of IgG from normal sera display TBII activity, suggesting that normal IgG also bind to human thyroid plasma membranes. Consequently, guinea pig fat cell membranes (FCM), which contain a high affinity receptor to TSH, were chosen as a receptor source. Initial experiments revealed that FCM failed to adsorb antimicrosomal antithyroid antibodies but were capable of adsorbing, with relatively high efficiency, the TBII activity in Graves' IgG. Additional experiments revealed that the TBII activity in normal IgG could be removed from FCM by several washes with buffer, whereas TBII activity in Graves' IgG could not. From these findings, a receptor purification technique was developed in which guinea pig FCM were first incubated with Graves' IgG, then washed with buffer, and then incubated with 0.1 M glycine-HCl, pH 3.0, to elute the adsorbed IgG. This method was applied to the isolation of Graves' disease-specific IgG from the serum of patients with this disorder. As judged from the IgG concentration required for 50% inhibition of [¹²⁵I] bovine TSH binding in both FCM and in human thyroid plasma membranes, about 40- to 150-fold enhancement of the TBII activity was achieved. Inhibition curves revealed close parallelism with those generated by the starting IgG. LATS activity and human thyroid stimulating activity in Graves' IgG were also enhanced by the receptor purification technique. Receptor-purified TBII activity was completely inhibited by the addition of rabbit antihuman IgG antibody, and a much lower anti-IgG concentration was required than was the case with the original TBII. Receptor-purified IgG contained no detectable antimicrosomal antibodies. The product obtained by the foregoing technique seems far closer to complete purification of a Graves'-specific IgG than any previously reported. It should prove to be useful as a starting material for efforts at purification and characterization and possibly for the development of aradioreceptor assay for Graves'-specific IgG.