Analytical Performance and Validation of a Bioassay for Thyroid-Blocking Antibodies

Diana, Tanja; Li, Yunsheng; Olivo, Paul D.; Lackner, Karl J.; Kim, Hannah; Kanitz, Michael; Kahaly, George J.

doi:10.1089/thy.2015.0447

Cited by 38 publications

(30 citation statements)

References 35 publications

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“…TSH-receptor blocking antibody bioassay. Serum TBAb activity was measured as described previously [12,13]. Briefly, a frozen vial of CHO-Mc4 cells (6Á7 3 10 4 cells/ well) was seeded and grown to a confluent cell monolayer in 96-well plates for 15-18 h at 378C, 5% CO 2 .…”

Section: Bioassays For Tsh-receptor Antibodiesmentioning

confidence: 99%

Prevalence and clinical relevance of thyroid stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease

Diana

Krause

Olivo

et al. 2017

Clinical and Experimental Immunology

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The prevalence and clinical relevance of thyroid stimulating hormone (TSH) receptor (TSHR) blocking antibodies (TBAb) in patients with autoimmune thyroid disease (AITD) was investigated. Serum TBAb were measured with a reporter gene bioassay using Chinese hamster ovary cells. Blocking activity was defined as percentage inhibition of luciferase expression relative to induction with bovine TSH alone (cut-off 40% inhibition). All samples were measured for TSHR stimulatory antibody (TSAb) and TSHR binding inhibiting immunoglobulins (TBII). A total of 1079 unselected, consecutive patients with AITD and 302 healthy controls were included. All unselected controls were negative for TBAb and TSAb. In contrast, the prevalence of TBAb-positive patients with Hashimoto's thyroiditis and Graves' disease was 67 of 722 (9·3%) and 15 of 357 (4·2%). Of the 82 TBAb-positive patients, thirty-nine (48%), 33 (40%) and 10 (12%) were hypothyroid, euthyroid and hyperthyroid, respectively. Ten patients were both TBAb- and TSAb-positive (four hypothyroid, two euthyroid and four hyperthyroid). Thyroid-associated orbitopathy was present in four of 82 (4·9%) TBAb-positive patients, with dual TSHR antibody positivity being observed in three. TBAb correlated positively with TBII (r = 0·67, P < 0·001) and negatively with TSAb (r = -0·86, P < 0·05). The percentage of TBII-positive patients was higher the higher the level of inhibition in the TBAb assay. Of the TBAb-positive samples with > 70% inhibition, 87% were TBII-positive. Functional TSHR antibodies impact thyroid status. TBAb determination is helpful in the evaluation and management of patients with AITD. The TBAb assay is a relevant and important tool to identify potentially reversible hypothyroidism.

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Section: Bioassays For Tsh-receptor Antibodiesmentioning

confidence: 99%

Prevalence and clinical relevance of thyroid stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease

Diana

Krause

Olivo

et al. 2017

Clinical and Experimental Immunology

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“…TSAb activity was reported as percentage of specimen-to-reference ratio (SRR%) (cutoff SRR% <140). Serum TBAb activity was measured as described previously [15,17]. Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (cutoff <40% inhibition).…”

Section: Tshr Stimulating and Blocking Antibody Bioassaysmentioning

confidence: 99%

“…All bioassay measurements were done in duplicate. Both bioassays have been validated [13][14][15], and the standardization of the TSAb bioassay was previously reported [16].…”

Section: Tshr Stimulating and Blocking Antibody Bioassaysmentioning

confidence: 99%

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Performance and Specificity of 6 Immunoassays for TSH Receptor Antibodies: A Multicenter Study

Diana¹,

Wüster²,

Olivo³

et al. 2017

Eur Thyroid J

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Background: The measurement of TSH receptor (TSHR) antibodies is warranted for diagnosis of Graves' disease (GD).Objective: The performance, detection sensitivity, and specificity of 6 TSHR immunoassays were compared. Methods: Two bioassays and 4 binding assays (Kronus, Immulite, Kryptor, Dynex) were compared in a dilution study performed in patients with autoimmune thyroid disease. Both bioassays were compared to 2 binding assays using stimulatory (M22) and blocking (K1-70) monoclonal antibody (MAb) mixtures. Results: Thirty samples from stimulatory (TSAb)-positive/blocking (TBAb)-negative patients with GD were diluted serially and measured in all assays. Samples were positive until dilution 1:2,187 in the TSAb bioassay, 1:81 in the Immulite (p < 0.002 vs. bioassay) and Kronus ELISA (p = 0.039) assays, and 1:27 in the Kryptor and Dynex ELISA (p < 0.001 vs. bioassay). Ten samples from TBAb-positive/TSAb-negative patients with GD or Hashimoto's thyroiditis were positive in all binding assays. None of the binding assays differentiated between TSAb and TBAb. Mixtures of 100% K1-70 (200 ng/ mL), 80% K1-70 + 20% M22, 60% K1-70 + 40% M22, 40% K1-70 + 60% M22, 20% K1-70 + 80% M22, and 100% M22 (20 ng/mL) tested positive in both Immulite (26.4, 20.2, 15.2, 10.5,6.3, 2.00 IU/L) and Kronus assays (27.1, 23.3, 19.3, 12.0, 5.7, 2.2 IU/L). These MAb mixtures were tested in the TBAb bioassay and showed 82, 61, 24 (negative), -26 (negative), -77 (negative), and -95% (negative) inhibition, respectively. Conclusions: The sample dilution study showed higher detection sensitivity for the TSAb bioassay, and the antibody mixture study demonstrated exclusive specificity of the bioassays over all automated and ELISA binding assays.

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“…It would appear that the signaling patterns utilized once TSHR is activated by TSIs are similar to those resulting from the actions of TSH but these are not identical (20,21). In addition to anti-TSHR antibodies that stimulate the receptor, others either block receptor activation or are neutral and assays are being developed to determine their levels in serum (22). It is important to stress that the same individual with GD can produce simultaneously both stimulatory and blocking antibodies, accounting perhaps for the rapid transition from hyperthyroidism to hypothyroidism observed in some patients.…”

Section: Characteristics Of Tshrmentioning

confidence: 99%

TSHR as a therapeutic target in Graves’ disease

Smith

2017

Expert Opinion on Therapeutic Targets

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Graves’ disease (GD) and its extrathyroidal manifestations such as thyroid-associated ophthalmopathy (TAO) are thought to result from abnormal activities of pathogenic antibodies mediated through the thyrotropin receptor (TSHR). At the core of GD is the loss of immune tolerance to TSHR and the generation of activating antibodies targeting the receptor. This then leads to the unregulated consequences of the antibody-mediated receptor activity in the thyroid and in connective tissues such as those inhabiting the orbit. While activating anti-TSHR antibodies can be detected in most individuals with GD, they appear to be absent or at exceedingly low levels in a subset of patients with the disease. Recent studies have disclosed the presence of antibodies that appear to be directed against the insulin-like growth factor-I receptor (IGF-IR). Their activities result in post-receptor signaling that is generally associated with IGF-IR. In this brief article, I have attempted to review the fundamental characteristics of the TSHR, its role in the pathogenesis of GD and TAO, and its relationship to IGF-IR. Strong evidence supports the concept that the two receptors form a physical and functional complex and that IGF-IR is required for some the down-stream signaling initiated through TSHR. Recently developed small molecules and monoclonal antibodies that block TSHR signaling as well as agents interrupting IGF-IR activation are also reviewed in the narrow context of their potential utility as therapeutics in GD and TAO. These agents possess the potential for offering more specific and better tolerated treatments for both hyperthyroidism and TAO, thus sparing patients exposure to toxic drugs, ionizing radiation and potentially hazardous surgeries.

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Analytical Performance and Validation of a Bioassay for Thyroid-Blocking Antibodies

Abstract: This TBAb bioassay exhibits excellent analytical performance and high level of reproducibility.

Cited by 38 publications

References 35 publications

Prevalence and clinical relevance of thyroid stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease

Prevalence and clinical relevance of thyroid stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease

Performance and Specificity of 6 Immunoassays for TSH Receptor Antibodies: A Multicenter Study

TSHR as a therapeutic target in Graves’ disease

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