Immunoprecipitation analysis of pathological autoantibodies in Graves' patients' sera using biotinylated human thyrotropin receptor labeled with 125I-neutravidiny.

Abstract

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.

• Authors: W B Minich, J D Weymayer, U Loos
• Journal: Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association (Exp Clin Endocrinol Diabetes) Vol. 107 Issue 8 Pg. 555-60 ( 1999) ISSN: 0947-7349 [Print] Germany
• PMID: 10612487 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Autoantibodies
• Iodine Radioisotopes
• Reagent Kits, Diagnostic
• Receptors, Thyrotropin
• neutravidin
• Avidin

Topics

• Autoantibodies (analysis)
• Avidin
• Biotinylation
• Graves Disease (immunology)
• HeLa Cells
• Humans
• Iodine Radioisotopes
• Precipitin Tests (methods)
• Reagent Kits, Diagnostic
• Receptors, Thyrotropin (analysis, immunology)