Immunoprecipitation analysis of pathological autoantibodies in Graves' patients' sera using biotinylated human thyrotropin receptor labeled with 125I-neutravidiny.
In the present article we describe a method for the direct immunoprecipitation analysis of pathological autoantibodies against TSH receptor (TSHR) in sera of patients with Graves' disease. For this purpose the fusion TSH receptor (TSHR-BIO-6HIS) was constructed. This fusion consists of the N-terminal 725 amino acids of the human TSHR linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of E. coli acetyl-CoA carboxylase (this domain directs the efficient posttranslational biotinylation of the protein) followed by 6 histidine sequence. TSHR-BIO-6HIS was produced in HeLa cells using recombinant vaccinia virus. The expressed receptor was complete active and was biotinylated with a high efficiency (about 90%). Biotinylated TSHR-BIO-6HIS was immobilized on Ni-NTA agarose and selectively labeled with a biotin binding protein-- 125I-neutravidin. The 125I-neutravidin labeled TSHR-BIO-6HIS, freed of the excess of nonbound radioactivity, was eluted from Ni-NTA agarose and used for the detection of pathological autoantibodies in 50 Graves' disease, 10 Hashimoto's disease, 10 insulin-dependent diabetes mellitus and 50 normal sera. 46 of 50 (92%) Graves' disease sera were positive in immunoprecipitation assay, as they have bound 125I-TSHR more effectively than the normal sera. There was a clear positive correlation between the immunoprecipitating activity and TSH-binding inhibiting activity of different Graves' sera (r = 0.69, P < 0.001). These findings pave the way for the development of a new practical assay, capable of detecting all pathological autoantibodies to the TSHR, particularly those which bind but do not affect the hormone-receptor interaction.
Minich
W B
WB
Department of Internal Medicine I, University of Ulm, Germany.
Weymayer
J D
JD
Loos
U
U
D001323:Autoantibodies / Q000032:analysis*; D001360:Avidin; D019921:Biotinylation; D006111:Graves Disease / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D011233:Precipitin Tests / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000276:immunology*
D001323:Autoantibodies; D007457:Iodine Radioisotopes; D011933:Reagent Kits, Diagnostic; D011989:Receptors, Thyrotropin; C093483:neutravidin; D001360:Avidin