Exp. Clin. Endocrinol. 100 (1992) 73-74
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Recombinant TSH-Receptor for Determination of TSH-Receptor-Antibodies M. Ludgate, S. Costagliola, D. Danguy, J. Perret and G. Vassart
Summary: We have expressed the complete coding sequence
menschlichen TSH-Rezeptors wurde in eukaryonten Zellen
of the human TSH-R in eucaryotic cells. Limiting dilution enabled us to select two clones, JPO9 and JP26, which have formed the basis of binding and bioassays respectively, for
exprimiert. Durch Anwendung der Limiting-Dilution-Technik konnten wir zwei Klone, JPO9 und JP26, selektionieren, die die Grundlage für Bindungs- und Bioassays zur Bestimmung von TSH-Rezeptor-Antikörpem bildeten. Die Ergebnisse des Bindungsassays zeigten eine gute Korrelation mit dem TRAK-Assay (Henning Berlin). Die Sensiti-
TSH-R antibodies.
Results obtained in the binding assay correlated well with those obtained in the TRAK (Henning Berlin) assay, while the bioassay was at least as sensitive as measurements made with FRTL5 or human thyroid cells. These cell lines provide a reliable source of human TSH-R which will be useful in routine diagnosis.
vität des Bioassays war mit der anderer Meßsysteme wie FRTL 5-Zellen und menschlicher Schilddrüsenzellen vergleichbar. Der Einsatz dieser Zellinien könnte auch in der Routinediagnostik von Nutzen sein.
Zusammenfassung: Die vollständige codierende Sequenz des
Introduction
TBAB which we have developed with these stably transfected cells.
The TSH-R is the target of autoantibodies which act as TSH agonists (TSAB) or antagonists (TBAB), the former resulting in hyperthyroidism and the latter the probable cause of hypothyroidism (Ludgate and Vassart, 1990). Currently a number of methods are in use which measure the inhibition of TSH binding (TBII) or the bioactivity of TSAB/TBAB and which thus play a role in diagnosis (Rees-Smith and McLachlan, 1988). There are
several disadvantages associated with
assays developed to date 1. frequently the TSH-R pre-
paration employed is of non-human origin; 2. there may be considerable heterogeneity in different batches of such preparations; 3. cell lines established to over-
come these problems may require prolonged culture before use in the assay.
The recent cloning and sequencing of the TSH-R (Libert et al., 1989; Parmentier et al., 1989) enabled us
to express it in eucaryotic cells (Perret et al., 1990). This paper will describe the assays for TBII and TSAB/
Methods Production of transfected cell lines CHO cells were cotransfected with a pSVL construct containing the complete coding sequence of the human TSH-R and
pSV2neo and selected by geneticin resistance to produce a mixed population of cells. These were cloned by limiting dilution and their accumulation of cAMP in response to TSH, forskolin and a TSAB were measured to select clones expressing high levels of the TSH-R (Perret et al., 1990).
Bioassay for TSABITBAB
This assay uses the JP26 cell line which has approximately 2000 TSR-R per cell. Duplicate wells of a microtitre plate were seeded with 50000 cells in Ham's F12 medium at 37°C overnight. Before the assay, medium was aspirated and the cells were washed in Hank's balanced salt solution without
Downloaded by: National University of Singapore. Copyrighted material.
Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles/Belgium
74
Exp. Clin. Endocrino!. 100 (1992) 1/2
NaCl. The cells were incubated in 200 tl of 1.5 mg/ml IgGs, from normals or patients, in 5.4mM KCI, 0.44 mM KH2PO4, 0.47 MgSO4, 0.35mM Na2HPO4, 0.95 mM CaC12, 0.1% glu-
TRAK assay performed according to the manufacturer's instructions (Henning Berlin).
cose, 2mM IBMIX, 20mM Hepes and 0.3% BSA for
Bindung assay for TBII This assay uses the JPO9 cell line which expresses approximately 90000 receptors per cell (Costagliola et al., 1992). Batches of solubilised TSH-R were prepared from the 40000 g membrane fraction from 10 confluent 9 cm petri dishes, about iO cells. This was resuspended in 1 ml of 75 mM Tris pH 7.5, 12.5mM MgC12, 0.6mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 sM leupeptin and 1 mM PMSF and added to 4 mls 1 0/ triton in 50mM NaC1 10mM Tris in which it was
homogenised. The suspension was centrifuged at 100000 g, the supernatant providing the TSH-R preparation. The assay was performed in a total volume of 200 11, 50 ILl
of TSH-R, 50 t'1 of serum and 100 sl of radio-iodinated bovine TSR, the kind gift of Henning Berlin. Incubation was for 1 hour at room temperature, the reaction was stopped by adding 300 Ill of cold 50 mM NaCl, 10 mM Tris followed by 500 t'1 30% polyethylene glycol in 1 m NaCl. Tubes were centrifuged at 13000 g for 5 minutes and the supernatant completely aspirated. Gamma radiation in the pellet was counted.
The NSB was determined in the presence of 10 mt'/ml unlabelled bovine TSH. Results are expressed as a percentage of total binding calculated as: counts in the presence of test serum counts in the presence of normal pool sera.
>
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Recombinant TSH-Receptor for Determination of TSH-Receptor-Antibodies M. Ludgate, S. Costagliola, D. Danguy, J. Perret and G. Vassart
Summary: We have expressed the complete coding sequence
menschlichen TSH-Rezeptors wurde in eukaryonten Zellen
of the human TSH-R in eucaryotic cells. Limiting dilution enabled us to select two clones, JPO9 and JP26, which have formed the basis of binding and bioassays respectively, for
exprimiert. Durch Anwendung der Limiting-Dilution-Technik konnten wir zwei Klone, JPO9 und JP26, selektionieren, die die Grundlage für Bindungs- und Bioassays zur Bestimmung von TSH-Rezeptor-Antikörpem bildeten. Die Ergebnisse des Bindungsassays zeigten eine gute Korrelation mit dem TRAK-Assay (Henning Berlin). Die Sensiti-
TSH-R antibodies.
Results obtained in the binding assay correlated well with those obtained in the TRAK (Henning Berlin) assay, while the bioassay was at least as sensitive as measurements made with FRTL5 or human thyroid cells. These cell lines provide a reliable source of human TSH-R which will be useful in routine diagnosis.
vität des Bioassays war mit der anderer Meßsysteme wie FRTL 5-Zellen und menschlicher Schilddrüsenzellen vergleichbar. Der Einsatz dieser Zellinien könnte auch in der Routinediagnostik von Nutzen sein.
Zusammenfassung: Die vollständige codierende Sequenz des
Introduction
TBAB which we have developed with these stably transfected cells.
The TSH-R is the target of autoantibodies which act as TSH agonists (TSAB) or antagonists (TBAB), the former resulting in hyperthyroidism and the latter the probable cause of hypothyroidism (Ludgate and Vassart, 1990). Currently a number of methods are in use which measure the inhibition of TSH binding (TBII) or the bioactivity of TSAB/TBAB and which thus play a role in diagnosis (Rees-Smith and McLachlan, 1988). There are
several disadvantages associated with
assays developed to date 1. frequently the TSH-R pre-
paration employed is of non-human origin; 2. there may be considerable heterogeneity in different batches of such preparations; 3. cell lines established to over-
come these problems may require prolonged culture before use in the assay.
The recent cloning and sequencing of the TSH-R (Libert et al., 1989; Parmentier et al., 1989) enabled us
to express it in eucaryotic cells (Perret et al., 1990). This paper will describe the assays for TBII and TSAB/
Methods Production of transfected cell lines CHO cells were cotransfected with a pSVL construct containing the complete coding sequence of the human TSH-R and
pSV2neo and selected by geneticin resistance to produce a mixed population of cells. These were cloned by limiting dilution and their accumulation of cAMP in response to TSH, forskolin and a TSAB were measured to select clones expressing high levels of the TSH-R (Perret et al., 1990).
Bioassay for TSABITBAB
This assay uses the JP26 cell line which has approximately 2000 TSR-R per cell. Duplicate wells of a microtitre plate were seeded with 50000 cells in Ham's F12 medium at 37°C overnight. Before the assay, medium was aspirated and the cells were washed in Hank's balanced salt solution without
Downloaded by: National University of Singapore. Copyrighted material.
Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles/Belgium
74
Exp. Clin. Endocrino!. 100 (1992) 1/2
NaCl. The cells were incubated in 200 tl of 1.5 mg/ml IgGs, from normals or patients, in 5.4mM KCI, 0.44 mM KH2PO4, 0.47 MgSO4, 0.35mM Na2HPO4, 0.95 mM CaC12, 0.1% glu-
TRAK assay performed according to the manufacturer's instructions (Henning Berlin).
cose, 2mM IBMIX, 20mM Hepes and 0.3% BSA for
Bindung assay for TBII This assay uses the JPO9 cell line which expresses approximately 90000 receptors per cell (Costagliola et al., 1992). Batches of solubilised TSH-R were prepared from the 40000 g membrane fraction from 10 confluent 9 cm petri dishes, about iO cells. This was resuspended in 1 ml of 75 mM Tris pH 7.5, 12.5mM MgC12, 0.6mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 sM leupeptin and 1 mM PMSF and added to 4 mls 1 0/ triton in 50mM NaC1 10mM Tris in which it was
homogenised. The suspension was centrifuged at 100000 g, the supernatant providing the TSH-R preparation. The assay was performed in a total volume of 200 11, 50 ILl
of TSH-R, 50 t'1 of serum and 100 sl of radio-iodinated bovine TSR, the kind gift of Henning Berlin. Incubation was for 1 hour at room temperature, the reaction was stopped by adding 300 Ill of cold 50 mM NaCl, 10 mM Tris followed by 500 t'1 30% polyethylene glycol in 1 m NaCl. Tubes were centrifuged at 13000 g for 5 minutes and the supernatant completely aspirated. Gamma radiation in the pellet was counted.
The NSB was determined in the presence of 10 mt'/ml unlabelled bovine TSH. Results are expressed as a percentage of total binding calculated as: counts in the presence of test serum counts in the presence of normal pool sera.
>
