Direct binding of thyrotropin receptor autoantibody to in vitro translatedthyrotropin receptor: A comparison to radioreceptor assay and thyroid stimulating bioassay

Graves' disease is characterized by the presence of autoantibodies to the t hyrotropin receptor (TSHR), which are pathogenic and responsible for diseas e activity. It is well recognized that the autoantibodies are heterogeneous and recognize a number of different conformational dependent epitopes on t he TSHR. In this study, we have extended our previous observations to study the interaction of Graves' disease autoantibodies with TSHR ectodomain pro duced by in vitro transcription and translation reaction. The specific acti vity 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 S-35-methionine and S-35- cysteine during the translation reaction. Addition of canine pancreatic mic rosomes to the translation mix showed that the glycosylation of TSHR ectodo main did not occur efficiently for the nascent receptor protein. In order t o determine the specificity and sensitivity of the improved assay with nong lycosylated 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 h istory of thyroid autoimmunity. With this large cohort of sera from Graves' disease and control individuals, 25% of Graves' disease sera immunoprecipi tated the dual labeled, in vitro transcribed and translated TSHR ectodomain , exceeding the 98th percentile of the control sera. There was no correlati on between the autoantibodies that immunoprecipitate the in vitro translate d TSHR ectodomain and those that inhibit iodinated TSH binding in the radio receptor assay and those with biological activity in a bioassay. The data a re consistent with the finding that a proportion of Graves' disease autoant ibodies can interact directly with TSHR ectodomain produced by in vitro tra nscription 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 TS HR immunoprecipitation on its own is unsuitable for diagnosis of Graves' di sease.