Increase of Thyroid Stimulating Activity in Graves' Immunoglobulin-G by High Polyethylene Glycol Concentrations Using Porcine Thyroid Cell Assay

Abstract

Cyclic adenosine monophosphate (cAMP) production during a 5-hour incubation using porcine thyroid cells (PTC) was stimulated significantly more by polyethylene glycol (PEG) 22.5% precipitated fractions (ppt frs) than by PEG 12.5% ppt frs from almost all Graves' sera. However, the thyrotropin (TSH) binding inhibition (TBI) activities of the PEG 12.5% and 22.5% ppt frs using porcine thyroid membranes were similar, and did not change in the 5-hour incubation. When the PEG 12.5% ppt fr from Graves' serum and the PEG 22.5% ppt fr from normal human serum (NHS) were coincubated, cAMP production was also stimulated as much as by the PEG 22.5% ppt fr from Graves' serum. When purified thyroid stimulating antibody (TSAb)-immunoglobulin G (IgG) and the PEG 22.5% ppt fr from NHS were coincubated, increased cAMP production was also observed, whereas bovine thyrotropin (bTSH) did not produce this effect. When purified TSAb-IgG and PEG solutions were coincubated, maximum increases in cAMP production (approximately 10-fold) with 5% PEG were found, whereas no increase was observed using bTSH. The stimulatory effect of high PEG concentrations on thyroid stimulating activity was observed by TSAb-IgG in salt-free or salt-containing medium (<0.15 mol/L NaCl concentration) but not by either TSAb-IgG conjugated to protein A-sepharose 4B or the inactivated TSAb-IgG by the treatment of 70 degrees C for 10 minutes. No stimulatory action by PEG was found with the thyroid stimulating substances such as GTPgammaS, forskolin, or pituitary adenylate-cyclase activating polypeptide (PACAP). The increased thyroid stimulating activity of Graves (IgG) at high PEG concentrations suggests the existence of some factors influencing the ability of TSAb to stimulate thyroid cells, although the exact mechanism remains to be clarified.

Clinical Significance of a Sensitive Assay for Thyroid-Stimulating Antibodies in Graves' Disease Using Polyethylene Glycol at High Concentrations and Porcine Thyroid Cells.

“…Consistent with this hypothesis, Kasagi et al (9) demonstrated by using porcine thyroid cells that cAMP response to the crude Ig fraction obtained from Graves' sera was stronger than that to the purified IgG. It was also demonstrated that coincubation of purified TSI with the PEG 22.5% ppt fr from normal human serum induced a significant increase in CAMP production (12). These observations have led to the conclusion that the assay with a high concentration of PEG is sensitive, specific and useful for the diagnosis and follow-up of Graves' disease after drug withdrawal.…”

“…According to their data, the PEG sediment proteins consisted of 27% albumin, 3.8% al-globulin, 7.2% a2-globulin, 12% n-globulin, and 49.5% r-globulin. Since Inui et al (12) demonstrated that the recovery of IgG in the PEG 12.5% ppt fr and PEG 22.5% ppt fr is more than 85%, the positive stimulatory effect is not dependent on the difference in the recovered IgG concentration.…”

“…Although the mechanism by which a high PEG concentration increases the thyroid stimulation activity by TSI remains unclear, Inui et al (12) proposed two hypotheses.…”

“…TSAb and sTSAb activities were determined according to the previously reported methods (9,12,13). Test sera (0.5 ml) were mixed with 1.5 ml of 18% or 30% PEG (6000) aqueous solution at 5°C, to make final PEG concentrations of 13.5% or 22.5%, respectively.…”

“…A crude f-globulin fraction obtained from sera by the polyethylene glycol (PEG) precipitation method has been hitherto used in the routine assay (9). More recently the Inui and Ochi group (12) reported that cAMP production during a 5-hour incubation with PTC was significantly augmented by using PEG 22.5% precipitated fractions instead of PEG 12.5% from almost all Graves' sera. In the present study, we demonstrated that TSI activities measured by the assay with the PEG 22.5% precipitation method (the PEG 22.5% assay with the detected antibody designated as sTSAb) was detected in almost all patients with untreated or recurrent active Graves' disease, whereas in the routine assay system with a crude r-globulin fraction obtained from sera precipitated with 13.5% PEG (the PEG 13.5% assay with the detected antibody designated as TSAb), thyroid stimulating antibody(TSAb) was detected in 85% of the patients with untreated Graves' disease and in 50% of the recurrent patients.…”