Re-I Tg had a markedly increased iodine content relative to Lo-I Tg, approaching 85% of Norm-I Tg (Fig. Tg, but not to Lo-I Tg. Serum antibodies from NOD.H2h4 mice with thyroiditis were poorly reactive to Lo-I Tg. To Naspm determine that these changes were due specifically to iodine content, Lo-I Tg was reiodinated reiodination (Re-I) of Tg. These data support the conclusion that humoral and cellular immune responses against Tg in the NOD.H2h4 mouse are dependent on the presence of iodine around the Tg molecule. Materials and methods Reagents and antibodies Monoclonal antibodies (mAbs) 42C3, 121B2 and 156A6 were supernatants of murine hybridomas raised against human Tg (huTg) but shown to be cross-reactive to mouse Naspm Tg (mTg) [11]. mAb 42C3 reactivity is usually iodine-dependent, as it does not recognize huTg from an endemic thyroid goitre with an undetectable iodine content [12]. Furthermore, the binding to huTg could be inhibited competitively by T4 or T3, but not non-iodothyroxine (T0) [12]. 121B2 and 156A6 binding to Tg is not I-dependent [3]. The antigenic specificity of mAb 42C3 has been described previously [3,12]. The reactivity of mAb 42C3 crosses species specificity, indicating that it recognizes a conserved epitope around the Tg molecule. Furthermore, it does not react to other iodinated control proteins such as bovine serum albumin iodinated iodination The iodine content of each preparation was assessed as previously described [5]. Briefly, 5 g of protein were hydrolysed in concentrated H2SO4. Organified iodine was displaced by 1 mM bromide and the change in OD405 in a ceric ammonium sulphate/sodium arsenate redox reaction plotted against a standard curve. For reiodination of Lo-I Tg, four iodobeads (Pierce, Rockford, IL, USA) were primed in 15 mM potassium iodide (Sigma) and added to 600 g of Lo-I or Norm-I Tg for 30 min at room temperature with continuous gentle agitation. Supernatants were dialysed against four changes of PBS [3], and the product was designated Re-I Tg. SDS-PAGE and Western blot Protein, 17 g, was loaded Rabbit polyclonal to PID1 into a 4C15% Tris-HCl gradient gel (Bio-Rad, Hercules, CA, USA) in denaturing, non-reducing loading buffer and run at 50 V for 35 h. Gels were either silver-stained (Pierce) or transferred onto Hybond-ECl nitrocellulose (Amersham Biosciences, Piscataway, NJ, USA) at 300 mA for 1 h at 4C, blocked overnight in PBS +5% non-fat dry milk at 4C, washed in PBS +005% Tween-20 and incubated with either 1 : 1000 gt -mTg or 1 : 500 with 42C3 hybridoma supernatant. Goat anti-mTg was identified with 1 : 500 rabbit anti-goat IgG-biotin (KPL, Gaithersburg, MD, USA) and 42C3 identified by goat anti-mouse IgG-biotin (Sigma). Both were revealed with 1 : 1000 extravidinCperoxidase (Sigma) and developed in 3,3-diaminobenzidine (Sigma). Enzyme-linked immunosorbent assay (ELISA) To evaluate the quantity of Tg in each preparation, microtitre plates were coated with each mTg at a concentration of 1 1 g/ml and probed using a goat antibody against mouse Tg, followed by washing and secondary incubation with polyclonal rabbit anti-goat Ig (KPL) secondary antibody conjugated to biotin followed by extravidinCperoxidase (Sigma). 3,3,5,5 Tetramethyl benzidine dihydrochloride (TMB) (KPL) was added as substrate and the plate was incubated for 5 min. The reaction was stopped with 018 M H2SO4 and the OD was read at 450 nm in a 96-well plate reader (Dynex, Chantilly, VA, USA). To determine the reactivity of monoclonal antibodies to the Tg preparations, microtitre plates were coated with Lo-I, Re-I and Norm-I as above and probed by mAbs 42C3, 121B2 and 156A6 supernatants diluted 1 : 100 in PBS followed by biotinylated goat -mouse Ig (Sigma) secondary, then extravidinCphosphatase (Sigma). Substrate 005). Untreated mice developed thyroid infiltrates over time with steadily increasing prevalence and severity. However, the Naspm prevalence of thyroid histopathology failed to become completely penetrant in untreated NOD.H2h4 animals, even in mice older than 1 12 months. The untreated mice that did progress to disease developed thyroid infiltrates with histopathological characteristics similar to the disease of iodine-fed NOD.H2h4 mice, including extensive mononuclear cell infiltration and thyroid follicle disruption (data not shown). In contrast, 8 weeks of dietary supplementation with 015% (w/v) sodium iodide in drinking water was sufficient to induce 100% penetrant.