Development of a luminescent bioassay for thyroid stimulating antibodies.

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating an tibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected rou tinely by measuring their ability to inhibit TSH binding to the receptor (T BII), which does not reflect their true biological activity. Current bioass ays which measure cAMP by RIA, are not suitable for routine use. We have de veloped a luminescent bioassay for TSAb, by introducing a cAMP responsive l uciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulu1 displays dose dependent TSH response detectable from 1 0 mu U/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. A n international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format f or automatic readout and 100 treated GD samples (50 TBII negative and 50 TB II positive) were tested, 73% being positive. In contrast only 4% of 79 con trol sera from individuals with Hashimoto's, non-thyroid autoimmunity or mu ltinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compare d in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescen t bioassay for TSAb, using unfractionated serum which is capable of high th roughput suitable for routine use.