title: Direct binding of thyrotropin receptor autoantibody to in vitro translated thyrotropin receptor: a comparison to radioreceptor assay and thyroid stimulating bioassay.
abstract: Graves' disease is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHR), which are pathogenic and responsible for disease activity. It is well recognized that the autoantibodies are heterogeneous and recognize a number of different conformational dependent epitopes on the TSHR. In this study, we have extended our previous observations to study the interaction of Graves' disease autoantibodies with TSHR ectodomain produced by in vitro transcription and translation reaction. The specific activity of the translated TSHR ectodomain has been increased by a log fold by adding an efficient ribosome binding Kozak sequence before the translation initiation codon as well as double labelling with 35S-methionine and 35S-cysteine during the translation reaction. Addition of canine pancreatic microsomes to the translation mix showed that the glycosylation of TSHR ectodomain did not occur efficiently for the nascent receptor protein. In order to determine the specificity and sensitivity of the improved assay with nonglycosylated TSHR ectodomain, we have studied 331 sera from Graves' disease patients and as controls 100 sera from patients with nonthyroid autoimmune disorders as well as sera from 200 normal control subjects with no family history of thyroid autoimmunity. With this large cohort of sera from Graves' disease and control individuals, 25% of Graves' disease sera immunoprecipitated the dual labeled, in vitro transcribed and translated TSHR ectodomain, exceeding the 98th percentile of the control sera. There was no correlation between the autoantibodies that immunoprecipitate the in vitro translated TSHR ectodomain and those that inhibit iodinated TSH binding in the radioreceptor assay and those with biological activity in a bioassay. The data are consistent with the finding that a proportion of Graves' disease autoantibodies can interact directly with TSHR ectodomain produced by in vitro transcription and translation. However, in contrast to the wide use of similar translation and immunoprecipitation assays to measure other autoantibodies for the diagnosis of autoimmune disorders, such as type 1 diabetes, the TSHR immunoprecipitation on its own is unsuitable for diagnosis of Graves' disease.
authors.affiliation: Department of Internal Medicine III, University of Leipzig, Germany. morgenthaler@brahms.de
authors.forename: N G
authors.initials: NG
authors.lastname: Morgenthaler
authors.forename: K
authors.initials: K
authors.lastname: Hodak
authors.forename: J
authors.initials: J
authors.lastname: Seissler
authors.forename: H
authors.initials: H
authors.lastname: Steinbrenner
authors.forename: I
authors.initials: I
authors.lastname: Pampel
authors.forename: M
authors.initials: M
authors.lastname: Gupta
authors.forename: A M
authors.initials: AM
authors.lastname: McGregor
authors.forename: W A
authors.initials: WA
authors.lastname: Scherbaum
authors.forename: J P
authors.initials: JP
authors.lastname: Banga
mesh_terms: D000818:Animals; D003062:Codon; D003545:Cysteine / Q000378:metabolism; D004285:Dogs; D000939:Epitopes / Q000032:analysis; D005260:Female; D006111:Graves Disease / Q000097:blood / Q000276:immunology*; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000097:blood*; D008297:Male; D008715:Methionine / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012016:Reference Values; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic
chemical_list: D003062:Codon; D000939:Epitopes; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D003545:Cysteine