Radiometric Measurement of Thyroglobulin-Antithyroglobulin Immune Complex in Human Serum

Abstract

A radiometric two-site assay for soluble thyroglobulin-antithyroglobulin immune complex (TgA) applicable to human serum has been developed. Rabbit anti-(human)IgG globulin and anti- (human)thyroglobulin (anti-Tg) were purified by affinity chromatography. When these antibeodies were labeled with ₁₂₅I, 45% and 62% could be bound to their corresponding antigens, IgG globulin and thyroglobulin, respectively. TgA was prepared by dissolving the immune precipitate (formed with human thyroglobulin [Tg] and human anti-Tg) in excess Tg and chromatographing the mixture on Sepharose 4B. On immunoelectrophoresis TgA migrated between IgG and Tg and formed precipitin lines against anti-IgG and anti-Tg. Serum unknowns were chromatographed on Sepharose 4B to separate TgA from free IgG; the heavy TgA eluated in the first fraction. Standard TgA or prepared unknown was first incubated in plastic cups precoated with anti-IgG. After washing cups (free Tg was removed at this step), bound TgA was identified by binding to cups of added ₁₂₅I-anti-Tg, the bound radioactivity being directly proportional to the amount of TgA present. Minimal detectable TgA was 0.4 ng/cup corresponding to a serum concentration of 16 ng/ml. The response curves of serial dilutions of serum eluate paralleled the standard curve. Coefficients of within assay variation ranged from 3.7 to 4.9% coefficients for between assay variation ranged from 20 to 28%. Preliminary data indicated that TgA was not detected in the sera of 10 normal subjects, but was detectable in 7 of 29 subjects (24%) with Graves' disease. The clinical significance of serum TgA levels remains to be determined by more extensive testing. The results indicate that a soluble immune complex, TgA, can be detected in serum with a high degree of sensitivity and reliability.