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Thyroid Stimulating Hormone (TSH) Receptor Antibodies. The family of autoantibodies known as TSH Receptor Antibodies is comprised of two members. Firstly, Thyroid Stimulating Immunoglobulins (TSI) which is characteristic of Graves disease, and is detected using an invitro bioassay. Secondly, Thyrotropin Binding Inhibitory Immunoglobulins (TBII) which compete, with thyrotropin, for binding to the receptor without necessarily leading to its stimulation. TBII is measured simply using a radioreceptor assay (Henning Trak Assay). TSI, however is detected using human TSH transfected host cells where bioactivity is measured. A detailed description of the TSI bioassay, as well as a comparison of the two methods, is provided in the following abstract. This abstract was presented at the 1998 Meeting of The American Thyroid Association in Portland Oregon. USE OF A STABLE CELL LINE, TRANSFECTED WITH THE HUMAN TSH RECEPTOR FOR IN VITRO BIOASSAY OF THYROID STIMULATING IMMUNOGLOBULINS (TSI). P. Ward, V. Tasevski, D. Howarth and B. Luttrell. Department of Endocrinology,Royal North Shore Hospital,Pacific Hwy, St Leonards NSW 2065 and Department of Nuclear Medicine, John Hunter Hospital, Newcastle, NSW AUSTRALIA. Thyroid Stimulating Immunoglobulins (TSI) in serum are indicative of autoimmune hyperthyroidism (Graves' Disease). These antibodies are measured by their stimulation of cAMP release upon interaction with the TSH receptor. Prior to 1993, primary cultures of human thyroid cells were used in this laboratory to measure the response (1), but the availability of human thyroid tissue was the major limitation. Other centres most commonly used a rat thyroid cell line (FRTL-5) even though the measurement of human antibodies with a rat-derived cell assay might introduce the possibility of errors due to species specificity. A new assay was devised using non-thyroidal cells (CHO cells) transfected with the cloned human TSH receptor (2) , kindly provided by G Vassart and J Dumont, Belgium, together with a highly sensitive inhouse RIA for cAMP (3). The procedure has been standardised against hTSH (WHO 84/703), and TSI samples were found to dilute in parallel with the TSH standard. There was good agreement between TSI activities as measured with the new CHO cell assay and the assay based on primary human thyroid cells (for a sample of 111 patients with suspected or treated Graves' disease a correlation r = 0.72 was obtained). A significant cAMP response was observed in 26 of 29 patients with untreated Graves' disease but not in 33 normal subjects. The new assay provides a different spectrum of antibody activity to receptor binding assays (thyrotrophin binding inhibitor immunoglobulin or TBII). For example, in a group of treated Graves' patients while thyrotoxic after disease recurrence we obtained a poor correlation (r = 0.31, n = 35) between TSI and TBII values obtained with a commercial kit (TRAK assay, Henning, Berlin). In conclusion, The TSH receptor-transfected CHO cell line affords an assay for TSI bioactivity, based on human material in a stable cell line. This avoids the batch to batch variation of primary thyroid cell cultures and also the potential problem of species specificity associated with the FRTL-5 line. 1. Grant, S., Luttrell, B., and Hales, I. 1985, Clin. Endocrinol. 23:3251. 2. Von Sande et al. 1992, Mol & Cell Endocrinol. 88 (R1-R5). 3. Luttrell, B.M. and Henniker, A.J. 1991, J.Biol Chem 266:21626-21630. Introduction: Thyrotrophin receptor antibodies (TRAb) is a generic term covering all antibodies that recognise the TSH receptor, their presence is indicative of autoimmune hyperthyroidism - Graves' Disease. Two methodologies are appropriate for the measurement of these circulating antibodies. The first of these is a radioreceptor assay in which serum immunoglobulins inhibit the binding of 125I-TSH to a solubilized receptor preparation. This activity has been functionally named as thyrotrophin binding inhibitory immunoglobulin (TBII). Thyroid Stimulating Immunoglobulins (TSI) are the bioactive class of the TRAb family and are measured in vitro by their stimulation of cAMP release upon interaction with the TSH receptor. Prior to 1993, primary cultures of human thyroid cells were used in this laboratory to measure TSI but the availability of human thyroid tissue was the major limitation. Other centres used the rat thyroid cell line (FRTL-5) despite the possibility of errors due to species specificity. A new assay was devised using CHO cells transfected with the human TSH receptor (CHO-R) (JP26) kindly provided by G. Vassart and J. Dumont (Belgium). The permanent cell line provides a convenient source of cells whilst avoiding the batch to batch variation that accompanies the use of primary cultures. Methods: CHO-R cells were routinely cultured in flasks containing Coons F12 medium, 2% FBS and 400 ug/mL geneticin. Prior to inclusion in the TSI bioassay, the cells were dispersed from the flask with trypsin and transferred to 96 well plates (1x105 cells per mL) and cultured until confluent in medium containing 10% FBS. Serum immunoglobulin, prepared using either saturated ammonium sulphate precipitation or polyethylene glycol (PEG) precipitation (final concentration of 15%), were incubated with the cells in a hypotonic salt solution containing 1mM 3-isobutyl-1-methylxanthine. The reaction was stopped with 0.05% sodium azide and the cell media was then analysed for the presence of cAMP by RIA. TBII was estimated as an index using the Brahms TRAK Assay kit (Berlin), the normal reference range established as >80. Results: Figure 1. Optimum conditions were established by culturing CHO-R cells in 96 well plates at a seeding density of 1x105 cells per mL for two days. The optimum incubation time for the TSI bioassay was determined as between 2 and 3 hours as measured by a times blank ratio. Figure 2. TSH dilution studies done in hypotonic salt solution and normal serum show clearly that the use of serum results in a less sensitive profile. There was good correlation, in terms of cAMP released, between sample preparation using PEG and ammonium sulphate precipitation. However, as is the case of TSH diluted in serum, samples prepared using PEG had a less sensitive dilution profile. The use of serum immunoglobulins obtained following (NH4)2SO4 precipitation and then dialysed against hypotonic salt gives optimum performance. Furthermore, samples prepared using (NH4)2SO4 precipitation did not cause the release of cAMP from non-receptor containing CHO cells. Figure 3. A comparison of TSI results obtained using primary human thyroid and CHO-R cells shows a correlation of r=0.71. Figure 4. In a TSI assay using primary human thyroid cells, Ig samples do not dilute in parallel with TSH. Within the CHO-R system, Ig samples dilute in parallel and the TSH dose response is enhanced. We speculate that this is due to a homogeneous population of TSH receptors. Figure 5. We have established a TSI bioassay that is standardized through the use of a reference preparation of human TSH in the range of 3.9 to 500uIU/mL. The normal reference range for TSI was established as <10 U/mL of TSI. The assay utilizes a highly sensitive in-house cAMP RIA. Figure 6. TBII correlated poorly with TSI overall and in a population of treated Graves' patients. Figure 7. A significant TSI result was observed in 26 of 29 patients with untreated Graves' disease but not in 33 normal patients. Data indicates that many Graves' patients continue to have significant levels of TSI following treatment. Conclusions: An assay has been developed in our laboratory for the measurement of TSI using CHO cells transfected with the TSH receptor. The method is broadly based on that of Grant (1) and utilises a highly sensitive in house cAMP RIA (3). The method developed is in good agreement with other publications (2,4) but importantly has been standardized against human TSH (WHO 84/703). Samples of serum immunoglobulin from patients with Graves' disease prepared using ammonium sulphate precipitation were found to dilute in parallel with TSH, a observation that was not seen when primary human thyroid cells were used in the bioassay. We speculate that this parallelism is due to a homogenous population of TSH receptors in the CHO-R line. The method is clinically relevant with 26 of 29 untreated Graves' patients having significant levels of TSI. Suprisingly, there was a poor correlation between TSI and TBII overall and in a population of treated Graves' patients. We are gathering more extensive information on these measures in a population of untreated Graves' patients. 1. Grant, S., Luttrell, B., and Hales, I. 1985, Clin. Endocrinol. 23:3251. 2. Von Sande et al. 1992, Mol & Cell Endocrinol. 88 (R1-R5). 3. Luttrell, B.M. and Henniker, A.J. 1991, J.Biol Chem 266:21626-21630 4. Vitti,P. etal. 1992, JCEM. 76:499-503 |