Fig. 2.

The epitope pattern of TgAb was assessed by measuring the inhibition of serum TgAb binding to Tg exerted by recombinant human TgAb-Fab in ELISA. Only sera with high titers (>100 U/ml) of TgAb were evaluated, because they were adequate to perform inhibition-binding experiments. Four TgAb-Fab to 4 different epitope regions (A, B, C and D) on Tg were employed. Wells coated with human Tg were incubated with serial dilutions of sera with high levels of TgAb in the presence of each TgAb-Fab. TgAb binding was detected with a horseradish peroxidase-conjugated antihuman IgG-Fc, which binds to IgG but not to Fab molecules. After the addition of substrate (o-phenylenediamine + H₂O₂), the optical density was read at 490 nm.