Abstract
In the present study, passive immunoneutralization was used as a means of neutralizing circulating thyroid hormones in vivo to study the response of the thyroid gland to such a physiological challenge. The mechanisms involved in the regulation of serum thyroid hormone levels and the effect of immunoneutralisation on lipid and carbohydrate metabolism, as reflected in serum glucose, cholesterol and triglyceride concentrations, were investigated. Murine monoclonal antibodies to thyroid hormones were produced in order to provide the required quantities of specific high-affinity antibodies for in vivo studies. Spleen cells of Balb/c mice immunized with either T3 or T4 were fused with Sp2 mouse myeloma cells. Enzyme-linked immunosorbent assays (ELISA) and the dot immunobinding assays were chosen for screening of the hybridoma cultures. Thirty clones secreting specific monoclonal antibodies to T3 and seven clones secreting T4 monoclonal antibodies were obtained. Monoclonal antibodies from two cell lines, MMT3. 1 and MMT4. 1, were isolated from ascitic fluid, purified and characterized with regard to titre, affinity, specificity and class. Sheep T3 and T4 polyclonal antibodies were also purified and characterized and proved to have high affinities and specificities. Binding characteristics of both monoclonal and polyclonal antibodies were investigated in vitro and polyclonal antisera were chosen for immunoneutralization study. In vivo immunoneutralisation studies were performed in male Wistar rats. Antibodies to T3, T4 or both were administered intraperitoneally and blood samples were collected at several time intervals. The affinity of the T3 polyclonal antiserum (8. 0 x 10-11mol/L) used in this study exceeded that of the binding capacity of T3 receptors of liver, kidney and heart; ranging between 1. 8 x 10 -9mol/L and 5 x 10-10mol/L depending on the experimental conditions, and the binding capacity of the amount of antibodies used amounted to more than 2500 times the total T3 and 20 times the total T4 in the rat serum. Since the antibodies administered interfered with the measurement of T3 and T4 by radioimmunoassay, serum samples collected from antiserum-treated animals were extracted with acid/ethanol. Serum T3 and T4, measured by a second antibody radioimmunoassay, were elevated in all experimental groups. TSH levels, on the other hand, remained at basal levels. This treatment with antibodies had no effect on total cholesterol concentrations whereas serum triglycerides were decreased and glucose levels were increased in all experimental groups. Long-term administration of T3 and/or T4 antiserum was also investigated. Animals were treated with a weekly dose of 0. 5ml antiserum and blood samples obtained, beginning from one day prior to antibody injection. Hormone levels as well as glucose, cholesterol and triglyceride showed no significant change throughout the study. Finally, the effect of long-term administration of thyroid hormones antibodies on several rat tissues including brain, pituitaries, thyroid, liver, kidneys and pancreas, all known sites of thyroid hormone action, was also investigated. The microscopic examination of sections of these tissues showed no abnormality compared with those from control rats.