Demonstration of thyroid stimulating activity within H chain fragments of TSAb-IgG by protease digestion and reduction

OBJECTIVE Whether or not the distribution of biologically active fragments in TSAb‐IgG molecules parallels antigen‐binding activity in other anti‐thyroidal antibodies was examined. DESIGN Both the thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) were examined by measuring the reduction in TSAb‐IgG followed by gel‐filtration on Sephadex G‐100. Two forms of IgG were separated from TSAb‐IgG by column chromatography using Protein L‐Sepharose (specifically binds to κ chain). The IgG(κ) was reduced with dithiothreitol (DTT) and the unbound fraction (UF) (the free heavy (H) chain) and the bound fraction (BF) (the free κ chain and the non‐reduced IgG(κ)) were separated using Protein L‐Sepharose. The Fab fragment was separated by Protein A‐Sepharose after papain hydrolysis of TSAb‐IgG, then separated into two Fab(κ) and Fab(λ) forms by Protein L‐Sepharose. The Fd fragment (or fragment containing Fd) was prepared from Fab(κ) by DTT reduction followed by Protein L column. RESULTS The free H chain fraction showed TS and TBI activity, but neither anti‐thyroglobulin (Tg) nor anti‐thyroid peroxidase (TPO) antibody activities. The free light (L) chain was not biologically active. Similar TS and TBI activities were found not only in IgG(κ) and IgG(λ) but also in the Fab(κ) and Fab(λ) fractions. Fd fragment that was not contaminated with free κ chain had TS and weak TBI activities. CONCLUSIONS The thyroid stimulating activity in 5 TSAb‐IgG samples was found in IgG (κ), IgG (λ), Fab (κ), Fab (λ), H chain and Fd separated by papain digestion and reduction. These results showed that TSAb was polyclonal and that the Fd fragment was important as the biologically active site.