Iodination of human thyroglobulin (Tg) alters its immunoreactivity. II. Fine specificity of a monoclonal antibody that recognizes iodinated Tg

Abstract

In a previous investigation, we found that murine MoAb 42C3, raised against human Tg, recognized Tg differently depending upon its level of iodination of Tg. A possible explanation for this finding is that iodine is directly involved with the specific epitope recognized by MoAb 42C3. In the present study, we report that the binding of MoAb 42C3 to iodinated Tg is inhibited by T4, T3, reverse T3 (rT3), triiodothyroacetic acid (triac), diiodothyronine (T2), diiodotyrosine (DIT), but not by thyronine (TO) or tyrosine. The order of inhibition of these iodinated compounds is T4 > T3 > rT3 > triac > T2 > DIT. The MoAb 42C3 does not have the same specificity as the T3, T4-receptor since the order of binding of these iodinated compounds on the receptor differed from the order of their inhibition of this MoAb. Monoclonal antibody 42C3 also recognized non-iodinated Tg that was subsequently iodinated in vitro. It failed to recognize another protein, bovine serum albumin, that was iodinated in vitro by the same method. These results suggest that iodinated tyrosines and thyronines determine the binding specificity of MoAb 42C3. The inhibitory effects of these compounds on MoAb 42C3 depend on their iodine content as well as location of iodine in the aromatic ring.

Fig. 1

Chemical structure of iodinated tyrosines and thyronines.

Fig. 2

Western immunoblot pattern of binding of MoAb 42C3 and anti-bovine serum albumin (BSA) with normal Tg (N-Tg), I^Neg-Tg, I⁺-Tg, BSA and I⁺-BSA. Thirty micrograms of each protein were analysed on 7.5% SDS–PAGE and the protein was transferred into nitrocellulose (NC) membranes. The membrane was either treated with MoAb 42C3 or anti-BSA. (a,b) The immunoblot pattern of these proteins with anti-BSA and MoAb 42C3, respectively. The molecular weights of protein standards are shown on the right. I^Neg-Tg, Tg with no detectable iodine from a patient with non-toxic goitre; I⁺-Tg, Tg with no iodine that was iodinated in vitro; I⁺-BSA, BSA that was iodinated in vitro.

Fig. 3

Inhibition of the immunoreactivity of binding of MoAb 42C3 to Tg by different concentrations of T4, T3, rT3, T2, T0, triiodothyroacetic acid and diiodotyrosine. Monoclonal antibody 42C3 was incubated with different concentrations of each compound at room temperature for 14 h. The T4 (thyroxine), T3 (triiodothyronine), rT3 (reverse T3), T2 (diiodothyronine), T0 (thyronine), diiodotyrosine, and triiodothyroacetic acid-treated antibody was then added to an ELISA plate coated with Tg. As control, untreated antibody was added to the plate. The amount of immunoglobulin bound to Tg was then measured by ELISA. Details of the ELISA are discussed in Materials and Methods. The figure shows inhibitory effect of each compound at concentrations of 0.0001 μg/ml to 100 μg/ml on binding of MoAb 42C3 to Tg in comparison with untreated (control) antibody.

Fig. 4

Inhibition of the immunoreactivity of binding of MoAb 133B1 to Tg by different concentrations of T4, T3, T2 and T0. Monoclonal antibody 133B1 was incubated with different concentrations of each compound at room temperature for 14 h. T4 (thyroxine), T3 (triiodothyronine), T2 (diiodothyronine) and T0 (thyronine)-treated antibody was then added to an ELISA plate coated with Tg. As control, untreated antibody was added to the plate. The amount of immunoglobulin bound to Tg was then measured by ELISA. Details of ELISA are discussed in Materials and Methods. The figure shows the inhibitory effect of each compound at concentrations of 0.0001 μg/ml to 100 μg/ml on binding of MoAb 133B1 to Tg in comparison with untreated (control) antibody.