A novel bioassay for thyroid-blocking immunoglobulins

Abstract

Background: Thyroid-blocking immunoglobulins (TBI) are present in 10%-15% of patients with autoimmune thyroid disease (AITD). TBI affect thyroid function. The analytical performance of a novel TBI bioassay was evaluated.

Methods: Sera from AITD patients were tested with a cell-based TBI reporter bioassay (Thyretain^®) with the expression of a luciferase transgene as readout and a new "Turbo™" TBI bioassay with a readout based on a cyclic AMP-activated luciferase. All samples were also run on two TSH-R binding immunoassays. A Passing-Bablok regression, a Bland-Altman plot, and user/lot comparisons were performed. In addition, dose-response curves for Turbo and Thyretain were fitted using serial dilutions, and half-maximal and 80% inhibitory concentrations (IC₅₀/IC₈₀) were compared.

Results: Of 1,011 unselected AITD patients, 131 patients (212 samples) were TBI positive. Of the 212 samples, 149 (70.3%), 47 (22%), and 16 (7.5%) were hypothyroid, euthyroid, and hyperthyroid, respectively. The three thyrotropin receptor antibody (TSH-R-Ab) assays were negative in 90 controls devoid of autoimmune thyroid disorders. In contrast, the Turbo cyclic adenosine 3',5'-monophosphate (cAMP) TBI, Thyretain TBI, and the binding assays detected TBI in 212 (100%), 168 (79%), and 138/180 (65%) samples, respectively (p< 0.001). Turbo highly correlated with thyroid function (p< 0.001). The percentage inhibition in both Turbo and Thyretain correlated with TSH-R-Ab binding assay positivity (both p< 0.001). The two bioassays correlated (r = 0.8, p< 0.001), and the Bland-Altman plot displayed no significant bias (0.24). Values scatter with slight systemic deviation between TBI mean values of 10%-50% inhibition, with higher Turbo than Thyretain results. Intra-assay validation demonstrated adequate precision with a very low coefficient of variation (average CV 5.4%) and lower CV with samples with a high inhibitory effect (CV_Average= 1.7% for a sample with 95% inhibition Thyretain). CV did not differ between users (p = 0.35) and lots (p = 0.121). The IC₅₀/IC₈₀ values were 1.55 ng/mL/3.48 ng/mL for Turbo and 6.76 ng/mL/18.46 ng/mL for Thyretain, respectively, demonstrating the markedly higher sensitivity of Turbo.

Conclusions: The novel, easy-to-perform, rapid, and reliable Turbo TSH-R blocking bioassay detected significantly more TBI than the established immunoassays, emphasizing its higher analytical performance and clinical utility in the management of patients with AITD.

Keywords: autoimmune thyroid disease; blocking TSH-R bioassay; homogeneous cAMP biosensor; thyroid-blocking immunoglobulins; thyrotropin receptor blocking antibodies.

Figure 1

Number of TBI-positive samples in various serological tests. N = 212 samples and 131 patients. Cutoff values: Turbo™ TBI >40 percentage inhibition; Thyretain^® TBI >34 percentage inhibition.

Figure 2

Turbo™ TBI Thyretain^® TBI TRAb immunoassay. ROC diagram of 302 samples (212 AITD and 90 controls) tested with Turbo™, Thyretain^® TBI and TRAb immunoassay. AUC (95% CI): Turbo™ 1,000 (0,986 - 1,000), Thyretain^® 0,950 (0,919 -0,972), and TRAb 0.952 (0.921-0.973).

Figure 3

, AITD; , control. Bland–Altman plot of Turbo™ and Thyretain^® TBI results.

Figure 4

, AITD; , control. Passing–Bablok regression of Turbo™ and Thyretain^® TBI results. The blue area marks a 95% confidence interval. N = 302 (221 subjects). Spearman’s Rho, 0.8; 95% CI, 0.75–0.84; p< 0.001. y = 12.878378 + 0.810811x.

Figure 5

(A) Dose–response curve by measuring a K1-70 dilution series with the Turbo™ TBI bioassay (IC₅₀ = 1.55 ng/mL, log (IC₅₀) = 0.19; IC₈₀ = 3.48 ng/mL, log (IC₈₀) = 0.54). (B) Dose–response curve by measuring a K1-70 dilution series with the Thyretain^® TBI bioassay (IC₅₀ = 6.76 ng/mL, log (IC₅₀) = 0.83; IC₈₀ = 18.45 ng/mL, log (IC₈₀) = 1.27).

Figure 6

, Average Turbo™ TBI values; , CV Turbo™ TBI; , Average Thyretain^® TBI values. Mean value of the Turbo™ TBI precision measurements with associated CV and the Thyretain^® TBI initial value of the respective sample. Cutoff values: Turbo™ TBI > 40 percentage inhibition; Thyretain^® TBI > 34 percentage inhibition. The average inhibition values are 59.45% (SD 54.15%) for user 1 and 54.15% (SD 16.72%) for user 2, with average CVs of 6% (SD 0.0492) for user one and 8% (SD 0.0735) for user 2 (p = 0.32). There was no statistical difference between Turbo™ and Thyretain^® values: Thyretain^® had an average inhibition of 60.8% (SD 22.02) and Turbo™ had an inhibition of 54.20% (SD, 16.92) (p = 0.27).

Figure 7

, Average values lot 1; , Average values lot 2; , CV lot 1; , CV lot 2. Presentation of the Turbo™ TBI mean values and CV. Cutoff values: Turbo™ TBI >40 percentage inhibition, Thyretain^® TBI >34 percentage inhibition. For the respective lots, the average inhibition values are 56.8% (SD 16.06%) for lot 1 and 49.0% (SD 15.69%) for lot 2, with no significant difference (p = 0.08). The CV is 7% (SD, 0.06) for lot 1 and 4% (SD, 0.03) for lot 2 (p = 0.34).