Micromethod of human thyrocyte cultures for detection of thyroid-stimulating antibodies and thyrotrophin

Abstract

Abstract. The activation of adenylate cyclase of human thyrocytes in primary cell cultures and the release of cAMP into the medium are used to detect thyrotrophin (TSH) and thyroid-stimulating antibodies (TSAb) in sera of patients with Graves' disease. Tissue digestion and cell dispersion are performed using a neutral protease of Bacillus polymyxa (Dipase II), which harvests more vital thyrocytes than does trypsin. The cells show an enhanced response to stimulation. The efficeincy of the cell preparation and cultivation is increased by using microtest plates instead of culture flasks. By this minimization only 0.6–1.8 × 106 cells are needed to evalute a single sample. Cyclic AMP concentrations are measured directly in the supernatant culture medium by a competitive protein binding assay with charcoal separation. The minimal detectable dose of bTSH is about 10 μU/ml. With increasing doses of bTSH cAMP concentrations rise to a peak at about 50 to 100 mU/ml, beyond which there is a gradual decrease of cAMP indicating negative cooperativity in the activating mechanisms. The 'long-acting thyroid stimulator' effect of TSAb is reflected by a protracted increase of cAMP to its maximal value. All 41 sera of hyperthyroid patients with Graves' disease were TSAb-positive, whereas sera of patients with toxic nodular goitre and of euthyroid controls were TSAb-negative.