Sensitive Assay to Detect Thyroid Stimulating Antibody (TSAb) in the Presence of Thyroid Stimulation Blocking Antibody (TSBAb) in Serum

The detection of thyroid stimulating antibody (TSAb) activity in the presence of thyroid stimulation blocking antibody (TSBAb) in Graves’ serum is difficult because TSBAb blocks TSAb activity. We recently demonstrated that polyethylene glycol (PEG) augments TSAb activity in porcine thyroid cells (PTC) assay. This PEG-induced augmentation makes it possible to develop a sensitive assay to detect TSAb in the presence of TSBAb. We studied the effects of PEG on TSAb- and TSBAb-activities in PTC using 4 different preparations of the samples; (1) crude IgG using PEG 22.5 % precipitated fraction (PF) from Graves’ serum (0.2 ml), (2) crude IgG using PEG 12.5 % PF, (3) serum (50 µl), and (4) serum (50 µl) in the presence of 5 % PEG (final). When the effects of PEG on TSAb activity using crude IgG were examined, PEG 22.5 % PF showed significantly higher TSAb activity than PEG 12.5 % PF as reported previously. The augmentative effect of PEG on TSAb activity was also observed by the addition of 5 % PEG to serum. We also demonstrated that PEG augmented TSAb-activities even in TSBAb-positive serum by two methods (crude IgG using PEG 22.5 % PF and the addition of 5 % PEG to serum). TSBAb activities were expressed by two calculation methods (A = [1 - (a - b)/(c - d) × 100] and B = [1 - (a - d)/(c - d) × 100], where a is cAMP produced in the presence of bTSH and patient’s IgG, b is cAMP produced in the presence of patient’s IgG, c is cAMP produced in the presence of bTSH and normal IgG, and d is cAMP produced in the presence of normal IgG). In the presence of TSAb, the values of A method were always higher than those of B method, since TSAb stimulated cAMP synthesis. We have developed two sensitive methods to detect TSAb even in the presence of TSBAb in serum using PEG; 1) incubation of crude IgG using PEG 22.5 % PF from serum (0.2 ml), and 2) co-incubation of 5 % PEG with test serum (50 µl).

Key words:

Thyroid Stimulating Antibody (TSAb) - Thyroid Stimulation Blocking Antibody (TSBAb) - cAMP - Graves’ Disease - Polyethylene Glycol (PEG)

References

• 1 Zakarija M, McKenzie M. Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves’ disease.  J Clin Invest. 1983;  72 1352-1356
• 2 Macchia E, Concetti R, Carone G, Borgoni F, Fenzi G F, Pinchera A. Demonstration of blocking immunoglobulin G, having a heterogenous behaviour, in sera of patients with Graves’ disease: Possible coexistence of different autoantibodies directed to the TSH Receptor.  Clin Endocrinol. 1988;  28 147-156
• 3 Miyauchi A, Amino N, Tamaki H, Kuma K. Coexistence of thyroid stimulating and thyroid blocking antibodies in a patient with Graves’ disease who had transient hypothyroidism.  Amer J Med. 1988;  85 418-420
• 4 Tamai H, Kasagi K, Takaichi Y, Takamatsu J, Komaki G, Matsubayashi S, Konishi J, Kuma K, Kumagai L, Nagataki S. Development of spontaneous hypothyroidism in patients with Graves’ disease treated with antithyroidal drugs: Clinical immunological and histological findings in 26 patients.  J Clin Endocrinol Metab. 1989;  69 49-53
• 5 Morita T, Tamai H, Oshima A, Mukuta T, Fukata S, Kuma K, Kumagai L F, Nagataki S. The occurrence of thyrotropin binding inhibitory immunoglobulin and thyroid stimulating antibodies in patients with silent thyroiditis.  J Clin Endocrinol Metab. 1990;  71 1051-1055
• 6 Takasu N, Yamada T, Sato A, Nakagawa M, Komiya I, Nakagawa Y, Asawa T. Graves’ disease following hypothyroidism due to Hashimoto’s disease: Studies of eight cases.  Clin Endocrinol. 1990;  33 687-698
• 7 Kasagi K, Hidaka A, Endo K, Miyamoto S, Takeuchi R, Misaki T, Sakahara H, Konishi J. Fluctuating thyroid function depending on the balance between stimulating and blocking types of TSH receptor antibodies: a case report.  Thyroid. 1993;  3 315-318
• 8 Michelangeli Y P, Poon C, Topliss D J, Colman P G. Specific effect of radioiodine treatment on TSAb and TBAb levels in patients with Graves’ disease.  Thyroid. 1995;  5 171-176
• 9 Inui T, Kouki T, Yamashiro K, Hachiya T, Ochi Y, Kajita Y, Sato Y, Nagata A. Increase of thyroid stimulating activity in Graves’ IgG by high polyethylene glycol concentrations using porcine thyroid cell assay.  Thyroid. 1998;  8 319-325
• 10 Yamashiro K, Takasu N, Kouki T, Inui T, Ochi Y, Kajita Y, Sato Y, Nagata A. Augmentation of thyroid stimulating antibody-stimulated cyclic adenosine monophosphate response by polyethylene glycol, polyvinyl alcohol and dextran; high sensitive porcine thyroid cell assay thyroid-stimulating antibody assay.  Thyroid. 1998;  9 263-271
• 11 Yamashiro K, Kouki T, Takasu N, Inui Y, Ochi Y, Kajita Y, Sato Y, Nagata A. Mechanism of the augmentative effect of high polyethylene glycol (PEG) concentrations on the tyroid stimulating activity in TSAb-IgG using porcine thyroid cell (PTC) assay.  Endocr Res. 1999;  25 67-76
• 12 Ochi Y, Inui T, Kouki T, Yamashiro K, Takasu Y, Kajita Y, Sato Y, Nagata A. Clinical usefulness of TSAb assay with high polyethylene glycol concentrations.  Horm Res. 1999;  51 142-149
• 13 Kamijo K, Nagata A, Sato Y. Clinical significance of a sensitive assay for thyroid-stimulating antibodies in Graves’ disease using polyethylene glycol at high concentrations and porcine thyroid cells.  Endocr J. 1999;  46 397-403
• 14 Kouki T, Inui T, Yamashiro K, Hachiya T, Ochi Y, Kajita Y, Takasu N, Sato A, Nagata A. Demonstration of fragments with thyroid stimulating activity from thyroid stimulation blocking antibodies-IgG molecules by papain digestion.  Clin Endocrinol. 1997;  47 693-698
• 15 Takeda K, Takamatsu J, Kasagi K, Sakane S, Ikegami Y, Isotani H, Majima T, Majima M, Kitaoka H, Iida Y, Ikekubo K, Konishi J, Mozai T. Development of hyperthyroidism following primary hypothyroidism: A case report with changes in thyroid-related antibodies.  Clin Endocrinol. 1988;  28 341-344
• 16 Chiovato L, Vitti P, Bendinelli G, Santani F, Fiore E, Capaccioli A, Tonacchera M, Mammoli C, Ludgate M, Pinchera A. Detection of antibodies blocking thyreotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor.  J Endocrinol Invest. 1994;  17 809-816
• 17 Kasagi K, Nagata A, Yoshimura H, Misaki T, Sato Y, Konishi J. Development and evaluation of a thyroid stimulation blocking antibody (TSBAb) assay using dispersed porcine thyroid cells. The proceedings of 71st Annual Meeting of the American Thyroid Association (abstract), 1998: 92
• 18 Konishi J, Iida Y, Endo K, Misaki T, Nohara Y, Matuura N, Mori T, Torizuka K. Inhibition of thyrotropin-induced adenine 3’5’-monophosphate increase by immunoglobulin from patients with primary myxedema.  J Clin Endocrinol Metab. 1983;  57 544-549
• 19 Inomata H, Sasaki N, Tamaru K, Ushiku H, Nimi H, Nakajima H. Relationship between potency of blocking type thyrotropin-binding inhibitor immunoglobulin in three women with primary myxedema and thyroid function of their neonates.  Endocr Jap. 1986;  33 353-359
• 20 Yokoyama N, Izumi M, Katamine S, Nagataki S. Heterogeneity of Graves’ immunoglobulin G; comparison of thyrotropin receptor antibodies in serum and in culture supernatants of lymphocytes transformed by Epstein-Barr virus infection.  J Clin Endocrinol Metab. 1987;  64 215-218
• 21 Matsui I, Sakata S, Ogawa T, Takuno H, Sarui H, Komaki T, Manshouri T, Atassi Z. Biological activities of rat antisera raised against synthetic peptide of human thyrotropin receptor.  Endocr J. 1993;  40 607-612
• 22 Hidaka Y, Guimaraes V, Soliman M, Yanagawa T, Okamoto Y, Quintans J, DeGroot L J. Production of thyroid-stimulating antibodies in mice by immunization with T-cell epitopes of human thyrotropin receptor.  Endocrinology. 1995;  136 1642-1647
• 23 Zhu Y, Portmann L, Denereaz L, Lemarchand-Beraud T. Simultaneous assay for three types of thyrotropin receptor antibody activities using FRTL-5 cells in patients with autoimmune thyroid disease.  Eur J Endocrinol. 1994;  131 359-368
• 24 Ochi Y, Yoshimura M, Hachiya T, Miyazaki T. Immunological studies on LATS-immunoglobulin by the reaction with Staphylococcal Protein A.  Endocrinol J. 1976;  23 183-186
• 25 Morgenthaler N G. New assay systems for thyrotropin receptor antibodies.  Curr Opin Endocrinol Diabetes. 1999;  6 251-260