Measurement of thyroid stimulating immunoglobulins in a new cell line transfected with a functional human TSH receptor (JPO9 cells), compared with an assay using FRTL-5 cells.
OBJECTIVE
Most bioassays used to measure thyroid stimulating antibodies (TSAb) rely on the ability of patient sera to generate cAMP in cultured cells of non-human origin. The cell line most commonly used has been the rat thyroid cell line, FRTL-5. Recently, a new cell line (JPO9) possessing a transfected human TSH receptor has been developed. The aim of this study was to evaluate the JPO9 cells by comparing them with the widely used FRTL-5 cells, using the method of 3H-adenine incorporation for the direct measurement of intracellular cAMP.


METHODS
The ability of patient sera to generate cAMP production in JPO9 and FRTL-5 cells was used as the index of thyroid stimulation. The specificity of the assay was determined using sera from patients with autoimmune thyroid and non-thyroid autoimmune diseases.


METHODS
We studied sera from 28 patients with newly diagnosed Graves' disease, 72 patients with Graves' disease at various stages of treatment, 8 patients with Hashimoto's thyroiditis, 40 patients with a variety of non-thyroid autoimmune disease and 42 control subjects.


METHODS
The intracellular 3H-cAMP generated in JPO9 cells following preincubation with 3H-adenine and subsequent incubation with patient or control sera, was used as a measure for TSAb. The results obtained with these cells were compared to those obtained with the widely used FRTL-5 cells and they were also correlated with the measurement of TSH binding inhibitory immunoglobulins by a conventional radioreceptor assay.


RESULTS
JPO9 cells were more sensitive and had a wider range of response to bovine TSH (bTSH) than had FRTL-5 cells (10(-7)-10(-2) U/ml compared to 10(-5)-10(-3) U/ml respectively). Unlike FRTL-5 cells, JPO9 cells respond well to unmodified serum and grow constitutively in the absence of TSH, thereby not requiring TSH deprivation prior to assay. The TSAb values obtained with both cell lines correlated well (r = 0.87). The JPO9 cells responded specifically to Graves' sera (23 out of 28 newly diagnosed patients were positive), whereas no patients with Hashimoto's thyroiditis and only 4 of 40 patients with non-thyroid autoimmune diseases gave low positive results for the presence of TSAb.


CONCLUSIONS
The JPO9 cells provide similar diagnostic information to FRTL-5 cells in patients with autoimmune thyroid disease. However, because they are more sensitive, grow faster, have less fastidious growth requirements and respond to unextracted sera, compared to FRTL-5 cells, we conclude that the JPO9 cells are preferable for the measurement of TSAb.
Michelangeli
V P
VP
Department of Diabetes and Endocrinology, Royal Melbourne Hospital, Parkville, Australia.
Munro
D S
DS
Poon
C W
CW
Frauman
A G
AG
Colman
P G
PG
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001323:Autoantibodies / Q000032:analysis*; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005069:Evaluation Studies as Topic; D006111:Graves Disease / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics; D013972:Thyrotropin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D000906:Antibodies; D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP