Isolation of radiochemically pure I-125-labeled human thyrotropin receptorand its use for the detection of pathological autoantibodies in sera from Graves' patients

We report a method for the purification and radioactive labeling of human T SH receptor (TSHR). The method is based on the construction of a fusion TSH R (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human T SHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino a cid C-terminal domain oi the biotin carboxyl carrier protein subunit of Esc herichia coli acetyl-CoA carboxylase (the C-terminal domain directs the eff icient posttranslational biotinylation of the protein). TSHR-Xa-BIO was pro duced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with I-125 using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pur e I-125-labeled TSHR specifically interacted with pathological autoantibodi es in the sera of patients with Graves' disease, and thus could be useful f or the detection of these autoantibodies by immunoprecipitation analysis.