Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor.
A clone of Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) was used to optimize an assay for thyroid-stimulating antibody (TSAb), measuring adenylate cyclase stimulation by purified immunoglobulin G from patients with Graves' disease. Optimal sensitivity to bovine TSH (1 mU/L) and TSAb was obtained using hypotonic buffer and measuring extracellular cAMP. In time-response experiments, TSAb stimulation was maximal after 2 h of incubation in hypotonic buffer. Under these conditions, a significant stimulation by Graves' immunoglobulin G was obtained with 33 of 35 (94%) samples from patients with untreated Graves' disease and with 21 of 23 (91%) from patients who relapsed after a course of antithyroid drugs. On the other hand, TSAb was detected in only 12 of 20 (60%) patients who were euthyroid during methimazole treatment and in 4 of 11 (36%) who were euthyroid after a course of antithyroid drugs. All samples from Graves' patients were also tested for TSAb activity on FRTL-5 cells. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation (r = 0.60; P < 0.0001). In particular, of the 58 patients with active Graves' disease (35 with untreated hyperthyroidism and 23 relapsed after methimazole), 43 (74%) were positive in both assays, 3 (5%) were negative in both, 11 (19%) were negative in FRTL-5 and positive in CHO-R, and 1 (1.7%) was negative in CHO-R and positive in FRTL-5. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture.
Vitti
P
P
Istituto di Endocrinologia, Università di Pisa, Italy.
Elisei
R
R
Tonacchera
M
M
Chiovato
L
L
Mancusi
F
F
Rago
T
T
Mammoli
C
C
Ludgate
M
M
Vassart
G
G
Pinchera
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001323:Autoantibodies / Q000032:analysis* / Q000502:physiology; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D006111:Graves Disease / Q000188:drug therapy / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000502:physiology; D018828:Immunoglobulins, Thyroid-Stimulating; D007700:Kinetics; D008713:Methimazole / Q000627:therapeutic use; D011989:Receptors, Thyrotropin / Q000235:genetics*; D013961:Thyroid Gland / Q000276:immunology / Q000378:metabolism; D013972:Thyrotropin / Q000494:pharmacology; D014162:Transfection
D001323:Autoantibodies; D007074:Immunoglobulin G; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D008713:Methimazole; D013972:Thyrotropin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases