title: Measurement of thyroid stimulating immunoglobulins using a novel thyroid stimulating hormone receptor-guanine nucleotide-binding protein, (GNAS) fusion bioassay.
abstract: Hyperthyroidism, defined by overproduction of thyroid hormones, has a 2-3% prevalence in the population. The most common form of hyperthyroidism is Graves' disease. A diagnostic biomarker for Graves' disease is the presence of immunoglobulins which bind to, and stimulate, the thyroid stimulating hormone receptor (TSHR), a G-protein coupled receptor (GPCR). We hypothesized that the ectopically expressed TSHR gene in a thyroid stimulating immunoglobulin (TSI) assay could be engineered to increase the accumulation of the GPCR pathway second messenger, cyclic AMP (cAMP), the molecule measured in the assay as a marker for pathway activation. An ectopically expressing TSHR-mutant guanine nucleotide-binding protein, (GNAS) Chinese hamster ovary (CHO) cell clone was constructed using standard molecular biology techniques. After incubation of the new clone with sera containing various levels of TSI, GPCR pathway activation was then quantified by measuring cAMP accumulation in the clone. The clone, together with a NaCl-free cell assay buffer containing 5% polyethylene glycol (PEG)6000, was tested against 56 Graves' patients, 27 toxic thyroid nodule patients and 119 normal patients. Using receiver operating characteristic analysis, when comparing normal with Graves' sera, the assay yielded a sensitivity of 93%, a specificity of 99% and an efficiency of 98%. Total complex precision (within-run, across runs and across days), presented as a percentage coefficient of variation, was found to be 7·8, 8·7 and 7·6% for low, medium and high TSI responding serum, respectively. We conclude that the performance of the new TSI assay provides sensitive detection of TSI, allowing for accurate, early detection of Graves' disease.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108-1221, USA. michael.l.pierce@aruplab.com
authors.forename: M
authors.initials: M
authors.lastname: Pierce
authors.forename: R
authors.initials: R
authors.lastname: Sandrock
authors.forename: G
authors.initials: G
authors.lastname: Gillespie
authors.forename: A W
authors.initials: AW
authors.lastname: Meikle
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006111:Graves Disease / Q000097:blood / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006980:Hyperthyroidism / Q000097:blood / Q000235:genetics / Q000378:metabolism; D018828:Immunoglobulins, Thyroid-Stimulating / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013961:Thyroid Gland / Q000378:metabolism
chemical_list: D018828:Immunoglobulins, Thyroid-Stimulating; D043562:Receptors, G-Protein-Coupled; D011989:Receptors, Thyrotropin; D011092:Polyethylene Glycols; D000242:Cyclic AMP; D019204:GTP-Binding Proteins