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2005 Georgia Veterinary Scholars

Georgia Veterinary Scholar	Faculty Mentor
David Wellington Tuskegee University Class of 2007	Dr. Duncan Ferguson

An immunoassay for Serum Rat Thyroglobin.

David A. Wellington*, Zach F.Caffall, and Duncan C. Ferguson. Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30606.

The physiological function of thyroglobin (Tg), a globular iodine-containing glycoprotein that occurs in the thyroid gland, is to provide the substrate for synthesis of the thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). The measurement of serum concentrations of thyroglobulin in human and rat sera have been shown to be proportionate to thyroid mass and secretory activity, both influenced by the average circulating thyrotropin (TSH) concentration. Furthermore, when iodide and iodothyronine content of the thyroid gland is examined, values are best normalized for glandular Tg content. In evaluating the thyroid-perturbing effects of environmental contaminants such as perchlorate and polychlorinated biphenyls (PCBs), we sought to develop an immunoassay for rat Tg. We chose to validate a double antibody immunoassay initially in enzyme-linked immunosorbant assay (ELISA) format. In our laboratory, 3 rabbit polyclonal antisera to feline Tg were available for evaluation, but no commercial purified rat Tg standard was available. Therefore, we purified rat thyroglobin extracted from fifty-thyroid glands of normal Sprague-dawley rats. The glands were dissected from the tracheal structure and surrounding tissues. The thyroids were minced and 1.0 ml of cold 0.15 M NaCl-0.0035 M phosphate-buffered saline (PBS), pH 7.0 was added. The total mixture was subsequently homogenized by a Potter-Elvehjem homogenizer. The homogenate was then centrifuged at 16,000 rpm for thirty-minutes at 4 °C to remove cellular debris. Following filtration, the supernate (4 ml) was treated with an equal amount of a saturated ammonium sulfate solution, and the resulting precipitate was centrifuged, redissolved in PBS, and extensively dialyzed against three changes of this buffer. The resulting solution was chromatographed on a 35 x 2.5-cm Sephadex G-200 column using PBS as the eluent, and fractions consistent with Tg molecular weight of 670,000 were collected This antigen will be tested initially in a sandwich ELISA format with purified immunoglobulin from one of the antisera coated on the plate, and immunoglobulin from a second antisera biotinylated, with detection by streptavidin- horse radish peroxidase enzyme (ELISA) or iodinated streptavidin immunoradiometric (IRMA) detection. This immunoassay will become an important tool in modeling the impact which environmental contaminant compounds have on hypothalamic-pituitary-thyroid axis of a model species, the rat.

[Supported by Environmental Protection Agency grant]