Available online 5 February 2023, 101744
Author links open overlay panelBioassays using animal models were essential tools in the discovery of thyrotropin and in enhancing our understanding of the physiology of the pituitary-thyroid axis. These same bioassays were also instrumental in the discovery of autoantibodies to the thyrotropin receptor (TSH-R-Ab) and in identifying their role in the pathophysiology of Graves’ disease. The development of cell-based bioassays led to further advances in our knowledge of the functional activity of TSH-R-Ab and to the discovery that TSH-R-Ab can be either thyroid-stimulating or thyroid blocking, and that they occur in other types of autoimmune thyroid diseases (AITD) besides Graves’ disease. More recently, TSH-R-Ab bioassays have been advanced from research tools to clinical laboratory tests. Whereas TSH-R-Ab can be measured with competitive-binding immunoassays, these assays do not provide information on the functional activity of TSH-R-Ab. Bioassays, in contrast, can differentiate between the stimulatory or blocking activity of TSH-R-Ab which provides clinically useful information that can inform the management of patients with AITD. The clinical use of TSH-R-Ab bioassays, however, has been limited to-date by their inherent complexity and long turn-around-time. Recent advances in biosensors have been applied to the development of TSH-R-Ab bioassays that are rapid and simple to perform. We now are entering an era in which bioassays for TSH-R-Ab can be measured routinely by virtually any clinical laboratory.
Section snippetsINTRODUCTIONA biological assay, or bioassay, is an analytical method used to measure the functional activity of a molecule on living organisms, tissue, or live cells. Bioassays are widely used to screen for potential hazardous chemicals in contaminated soils, wastewater, or foods, and are an important tool in the development of new drugs [1], [2], [3], [4], [5], [6], [*7]. One of the first described bioassays was a bioassay for diphtheria antitoxin developed by Paul Ehrlich ([8]. In medicine, bioassays
THYROTROPIN RECEPTORThe TSH-R is found on thyrocytes and the cells of other tissues and is a member of the G protein-coupled receptors (GPCRs), the largest known superfamily of cell surface receptors [20]. TSH, produced by the pituitary gland, binds to the TSH-R and activates signal transduction pathways that promote thyrocyte proliferation and production of thyroid hormone. As with all GCPRs, conformation changes in TSH-R occur upon binding of TSH or other agonists, which initiates a signaling cascade in the
AUTOIMMUNE THYROID DISEASEAutoimmune thyroid diseases (AITD) are common causes of both hyperthyoidism and hypothyroidism and are characterized by the presence of autoantibodies to a number of thyroid antigens such as thyroid peroxidase (TPO) and TSH-R [25]. Although anti-TPO antibodies are highly prevalent in patients with AITD, these antibodies are not thought to be directly involved in the pathogenesis of AITD. In contrast, TSH-R-Ab are uniquely and directly involved in the pathophysiology of certain types of AITD. In
TERMINOLOGYThere are a variety of terms used to describe the different types of TSH-R-Ab. TSH-R antibody (TRAb) refers to any type of autoantibody specific to the TSH-R, but it is commonly used in reference to antibodies detected in an immunoassay. Most immunoassays today use a competitive-binding assay called TSH-R binding inhibitory immunoglobulin (TBII) assay. In contrast, bioassays measure either thyroid stimulatory antibodies (TSAb), also referred to as thyroid stimulating immunoglobulins (TSI), or
ANTI-TSH-R MONOCLONAL ANTIBODIESAnti-TSH-R monoclonal antibodies have been important in efforts to understand the role of TSH-R-Ab in pathogenesis of AITD. A number of murine anti-TSH-R monoclonal antibodies have been isolated and described, but the most important development in this area is the isolation of human monoclonal antibodies to the TSH-R from AITD patients [35], [36], [37], [38], [39], [40]. Ever since it has been known that TSH-R-Ab exhibit different functional activities, it has been suspected that certain
BIOASSAYS FOR TSH-R-ABA summary of the advances in the development of TSH-R-Ab bioassays is shown in Table 1. The first generation of TSAb bioassays used the same principles that had been used to measure TSH activity. These early TSAb bioassays used cultured dog thyroid cells, human thyroid cells and later a continuous rat thyrocyte cell line (FRTL-5) [49], [50], [51]. Concerns about differences between the human and rat TSH-R lead to the use of cell lines transfected with the gene for human TSH-R. The most common
LATEST GENERATION OF BIOASSAYSRecently, novel biosensors have been developed that allow for real-time measurement of cAMP dynamics inside live cells and thus can be used to provide a quantitative measurement of GPCR activation [*68], [69], [70], [71]. These biosensors allow for simple, homogeneous, high-throughput screening assays for cAMP and thus are excellent tools in both academic and pharmaceutical laboratories. A live-cell TSAb bioassay that uses a cyclic nucleotide-gated calcium channel and aequorin was recently
BIOASSAYS FOR THYROID-BLOCKING AUTOANTIBODIES (TBAB)Thyroid-blocking activity is caused by TSH-R-Ab that block the stimulatory action of TSH. Initial bioassays for the measurement of thyroid-blocking activity were based on the same cell-based systems as used for TSAb, but they detected the ability of patient antisera to block TSH or TSAb-stimulated cAMP levels [74], [75], [76], [77], [78], [79]. Other bioassays for detecting TBAb were based on the inhibition of the TSH-mediated proliferation of FRTL-5 cells using tritiated nucleoside
CONCERNS ABOUT TSAB AND TBAB BIOASSAYSA number of concerns have been raised about the accuracy and relevance of bioassays in general, and TSAb/TBAb bioassays, in particular. The first concern relates to the fact that the original bioassays used thyroid cells and measured thyrocyte proliferation and hormone production. Thus, these bioassays could be thought to more closely reflect the in vivo activity of the antibodies. In order to streamline TSAb bioassays, non-thyroid cells, engineered to express human TSH-R, or mutant forms of
SUMMARYMethodologies available for bioassays have advanced significantly. The most recent generation of bioassays do not require laboratories to perform cell culture and the protocols are very straightforward and can be easily automated. This raises the possibility of routine use of selective bioassays in clinical laboratories. In the case of TSH-R-Ab bioassays, this is important because these bioassays provide results on the functional activity of TSH-R-Ab which has clinical utility and which can be
AcknowledgementsThe author wishes to acknowledge the helpful discussions with my many colleagues
DisclosurePDO consults for Quidel Corporation, San Diego, USA
PRACTICE POINTS OR TAKE-HOME MESSAGES•Bioassays continue to play an important role in medicine.
•Bioassays for TSHR-Ab have been important tools for understanding the pathogenesis of AITD.
•Bioassays for TSHR-Ab can determine the functional activity of TSHR-Ab
•Advances in bioassay methodology have improved significantly and TSHR-Ab bioassays are no longer complex, laborious, and slow.
•Routine use of TSHR-Ab
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