Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves'...

We describe a new method for the detection of different types of pathological autoantibodies against TSH receptor (TSHR) in Graves' patients sera by luminescent immunoprecipitation analysis. For this purpose three different chimeras composed of human TSHR and rat luteotropin/choriogonadotropin receptor (LH-CGR) were constructed, as was described previously (Tahara K, Ishikawa N, Yamamoto K, Hirai A, Ito K, Tamura Y, Yoshida S, Saito Y, Kohn LD. 1997 Thyroid 7:867-877). They were used in the immunoprecipitation reactions: (i) the wild type TSHR (for the detection of total TSHR autoantibodies), (ii) TSHR/LH-CGR chimera wherein TSHR amino acid residues 8-165 (epitopes for thyroid stimulating antibodies) are replaced by comparable LH-CGR residues, (iii) TSHR/LH-CGR chimera wherein TSHR amino acids 261-370 (epitopes for thyroid blocking antibodies) are replaced by comparable LH-CGR residues, and (iv) TSHR/LH-CGR chimera wherein TSHR amino acids 8-165 and 261-370 are replaced by comparable LH-CGR residues (for the detection of neutral TSHR autoantibodies). DNA encoding the N-terminal 725 (of 764) amino acids of wild type TSHR (or TSHR/LH-CGR chimera) was fused to the cDNA for the 550-amino acid firefly luciferase. The hybrid proteins were produced in HeLa cells using recombinant vaccinia viruses. All fusion proteins retained the enzymatic activity of firefly luciferase and TSHR-LUC interacted with TSH with the same affinity as wild type receptor. The luciferase tagged TSHR and TSHR/LH-CGR chimeras were used for the detection of different types of TSHR autoantibodies (i.e. total, neutral, thyroid stimulating and thyroid blocking) in 63 Graves' disease and 62 normal sera by immunoprecipitation analysis. The data demonstrated positive correlation between results of immunoprecipitation assay and results obtained using cAMP bioassay or assay for TSH binding inhibitory immunoglobulins in test sera.