Thyroid Stimulating Antibody Assay with Chinese Hamster Ovary Cells Expressing Human Thyroid Stimulating Hormone (TSH) Receptor; Optimization of Assay Condition / 대한내분비학회지

ABSTRACT

We investigated the optimal condition of thyroid stimulating antibody(TSAb) assay using Chinese hamster ovary cells transfected with cDNA of human TSH receptor(TSHr-CHO) stably expressing functional TSH receptors. The extracellular cAMP responses of TSHr-CHO cells to the stimulation of bTSH or Graves' IgG were observed in three different incubation media. Stimulation indices of extracellular cAMP were higher when sucrose containing NaCl-free isotonic Hank's balanced salt solution(HBSS)(media A)was used as incubation media than those of NaCl-free hypotonic HBSS(media B) or those of NaCl containing isotonic HBSS(media C). The incubation of TSHr-CHO cells in media B caused marked increase in the basal cAMP level without concomittant fold-increase in the stimulated cAMP level at various doses of bTSH and Graves' IgG. Decreasing the stimulation indices of extracellular cAMP, use of media B failed to detect TSAb activities in two TSAb-positive Graves' IgG tested. In case of media C, extracellular cAMP responses are poor at 0.001 and 0.1U/L of bTSH and at all doses of Graves' IgG tested(0.5, 1, 5g/L). The incubation of TSHr-CHO cells in media B caused significant increase in the number of trypan blue-stained, nonviable cells(5.7+-1.5, 7.6+-1.9 and 8.5+-1.6% at 1, 2 and 3h of incubation, respectively; p<0.01) comparing to those incubated in media A or media C(about 2-3% in both media). Those decrease in the viability of TSHr-CHO cells when incubated in hypotonic incubation media may explain the decrease in the stimulation index of extracellular cAMP with the use of media B in contrast to the case of FRTL-5 cells. TSAb assay of 87 consecutive fresh Graves' patients with TSHr-CHO cells using media A detected TSAb activities in 90%(78 patients) of them, and moreover TSAb activities showed significant positive correlation with the pre-treatment serum T_3 and free T_4 levels of those patients. We conclude that TSAb assay with TSHr-CHO cells is a sensitive and physiologically relevant assay system to measure TSAb activities merely through measurements of extracellular cAMP provided that the cells are incubated in NaCl-free isotonic incubation media.