Monoclonal Antithyroglobulin Antibodies Derived from Immunizations of Mice with Human Eye Muscle and Thyroid Membranes*

Abstract

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

Human fibrocytes coexpress thyroglobulin and thyrotropin receptor

“…This same group generated anti-Tg mAbs by immunizing mice with extraocular muscle membranes (30). More recently, Marinò et al (14) detected intact Tg by Western blot analysis and ELISA in orbital tissues from three patients with TAO.…”

Significance of Thyroid Stimulating Antibody and Long Term Follow Up in Patients with Euthyroid Graves' Disease.

“…As the histological evidence of autoimmunity, lymphocyte and macrophage infiltration and peptide factors such as IL-1a, TNFa, IFNy, TGF/3, PDGF and IGF-I, were detected in retroorbital tissues [41][42][43][44]. Anti-thyroglobulin antibody cross-reacted with human orbital tissue [45,46]. Wang et al [47] and Dillon et al [48] suggested antibody dependent cytotoxicity acting on human eye muscle cells.…”

Identification of Thyroglobulin in Orbital Tissues of Patients with Thyroid-Associated Ophthalmopathy

“…bodies to extraocular muscle membranes and attempts to detect Tg in tissue sections by immunohistochemical techniques produced negative results (29). In all these studies (14)(15)(16)(17)29) orbital tissues were obtained at autopsy from subjects with neither thyroid disease nor TAO.…”

“…In all these studies (14)(15)(16)(17)29) orbital tissues were obtained at autopsy from subjects with neither thyroid disease nor TAO. Because serum Tg levels in normal subjects and in patients without thyroid diseases are usually low or undetectable (30), it is likely that little, if any, deposition of Tg occurs in orbital tissues to detectable levels.…”