Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves' patients sera by luminescent immunoprecipitation analysis

We describe a new method for the detection of different types of pathologic al autoantibodies against TSH receptor (TSHR) in Graves' patients sera by l uminescent immunoprecipitation analysis. For this purpose three different c himeras composed of human TSHR and rat luteotropin/choriogonadotropin recep tor (LH-CGR) were constructed, as was described previously (Tahara K. Ishik awa N, Yamamoto K, Hirai A, Ito K, Tamura Y,Yoshida S, Saito Y. Kohn LD. 19 97 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 fo r thyroid stimulating antibodies) are replaced by comparable LH-CGR residue s. (iii) TSHR/LH-CGR chimera wherein TSHR amino acids 261-370 (epitopes for thyroid blocking antibodies) are replaced by comparable LH-CGR residues. a nd (iv) TSHR/LH-CGR chimera wherein TSHR amino acids 8-165 and 251-370 are replaced by comparable LH-CGR residues (for the detection of neutral TSHR a utoantibodies). DNA encoding the N-terminal 725 (of 764) amino acids of wil d type TSI-IR (or TSHR/LH-CGR chimera) was fused to the cDNA for the 550-am ino acid firefly luciferase. The hybrid proteins were produced in HeLa cell s using recombinant vaccinia viruses. All fusion proteins retained the enzy matic activity of firefly luciferase anti TSHR-LUC interacted with TSH with the same affinity as wild type receptor. The luciferase tagged TSHR and TS HR/LH-CGR chimeras were used for the detection of different types of TSHR a utoantibodies (i.e. total, neutral, thyroid stimulating and thyroid blockin g) in 63 Graves' disease and 62 normal sera by immunoprecipitation analysis . The data demonstrated positive correlation between results of immunopreci pitation assay and results obtained using cAMP bioassay or assay for TSH bi nding inhibitory immunoglobulins in test sera.