Measurement of thyroid-stimulating immunoglobulins by incorporation of tritiated-adenine into intact FRTL-5 cells: a viable alternative to radioimmunoassay for the measurement of cAMP.
OBJECTIVE
To evaluate the utility of 3H-adenine incorporation into intact rat thyroid epithelial cells (FRTL-5) as an alternative to radioimmunoassay for the measurement of cAMP following stimulation of these cells by serum thyroid-stimulating immunoglobulins from patients with Graves' disease.


METHODS
We determined the cAMP produced by FRTL-5 cells following incubation with serum from patients with a spectrum of autoimmune thyroid and other diseases using the 3H-adenine assay.


METHODS
We studied 27 patients with untreated Graves' disease, 10 with Graves' disease complicated by ophthalmopathy (all on antithyroid medication), 11 with Hashimoto's thyroiditis, five with multinodular goitre, one with thyroid carcinoma, 23 with type 1 diabetes mellitus, 19 with other autoimmune diseases and 10 controls.


METHODS
The 3H-cAMP produced in cells incubated with either bovine TSH (bTSH) or polyethylene glycol-precipitated serum immunoglobulins, was separated by sequential chromatography on Dowex and alumina columns, and counted. The thyroid-stimulating immunoglobulins index (3H-cAMP patient immunoglobulins/3H-cAMP control immunoglobulins) was calculated for each serum and considered positive if greater than 1.5 (+ve thyroid-stimulating immunoglobulins index, i.e. > 2 standard deviations above control). The thyroid-stimulating immunoglobulins index was correlated with measurement of thyrotrophin binding inhibitory immunoglobulins (TBII) by radioreceptor assay.


RESULTS
The 3H-adenine assay has a sensitivity of 10(-11) M bTSH with maximal stimulation at 10(-9) M bTSH (30-fold). Twenty-five of 27 patients (92%) with untreated Graves' disease and four of 10 patients with Graves' disease complicated by ophthalmopathy had +ve thyroid-stimulating immunoglobulin indices. The thyroid-stimulating immunoglobulins index in patients with untreated Graves' disease correlated with their TBII assay result (r = 0.63, P < 0.001). In addition, the index was negative in patients with Hashimoto's thyroiditis, multinodular goitre, thyroid carcinoma, and type 1 diabetes mellitus. Of the patients with other autoimmune diseases only one (a patient with systemic lupus erythematosis) had a +ve thyroid-stimulating immunoglobulin index. Direct comparison of cAMP measurement by 3H-adenine incorporation and commercial radioimmunoassay showed an equal sensitivity to both bTSH and Graves' immunoglobulins. After cell preparation, results are obtained more quickly with the 3H-adenine assay than with a cAMP radioimmunoassay (5 hours compared to 2 days), and far more cheaply than by commercial radioimmunoassays.


CONCLUSIONS
Measurement of thyroid-stimulating immunoglobulins using the incorporation of 3H-adenine into cAMP in FRTL-5 cells is sensitive, reproducible, rapid and specific. These features make this assay a viable alternative to RIA for the measurement of thyroid-stimulating immunoglobulins in patients with Graves's disease.
Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
P J
PJ
Leedman
A G
AG
Frauman
P G
PG
Colman
V P
VP
Michelangeli
D000225:Adenine / Q000378:metabolism*; D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis*; D004848:Epithelium / Q000276:immunology / Q000378:metabolism; D006111:Graves Disease / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D051381:Rats; D012680:Sensitivity and Specificity; D013959:Thyroid Diseases / Q000378:metabolism; D013961:Thyroid Gland / Q000276:immunology / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism*
D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D014316:Tritium; D000242:Cyclic AMP; D000225:Adenine