Development of More Sensitive Bioassay of TSAb Due to the Modification of Conventional Assay and its Measurement in M22-TRAb-Seronegative Graves' Patients

Research Article

Annals Thyroid Res. 2014;1(1): 5-10.

Development of More Sensitive Bioassay of TSAb Due to the Modification of Conventional Assay and its Measurement in M22-TRAb-Seronegative Graves' Patients

Keiichi Kamijo1* and Kazuyoshi Togashi2

1Kamijo Thyroid Clinic and Kamijo Thyroid Research Institute, Japan

2Yamasa Corporations, Japan

*Corresponding author: Keiichi Kamijo, Kamijo Thyroid Clinic and Kamijo Thyroid Research Institute, Sapporo, Hokkaido, Japan

Received: August 18, 2014; Accepted: Sep 11, 2014; Published: Sep 15, 2014

Abstract

Background: Graves’ disease (GD) is caused by thyrotropin (TSH) receptor antibodies (TRAb) that bind to TSHR and activate thyrocytes. The measurement of TRAb therefore has been used to assist in the diagnosis of GD, although approximately 3 % of GD patients were negative for M22-TRAb. This study evaluated a newly modified TSAb (referred as TSAb) assay using porcine thyroid cells which is characterized by more sensitive and rapid assay system compared with conventional TSAb assay.

Methods: First, we compared TSAb and conventional TSAb assay using porcine cells. In TSAb assay, sera were treated with dextran coated charcoal to remove intrinsic AMP and the incubation buffer contained 6% of polyethylene glycol to intensify the cAMP production of porcine cells due to TSAb stimulation. After cell lysis with TritonX-100, cAMP was measured with EIA.

Next, TSAb were measured using a modified TSAb assay in 17 M22-TRAbseronegative patients with untreated GD to investigate whether these cases have autoantibodies against the TSH receptor.

Results: The sensitivity and specificity of TSAb for GD and Painless Thyroiditis (PT) were 99.1% and 94.7%, respectively, at the optimal cut-off value of 120%. The positive predictive value of TSAb for GD was significantly higher than that of conventional TSAb (99.1% vs. 94.5%, p<0.05) and the negative predictive rate of PT by TSAb was also significantly higher than that of conventional TSAb (94.7% vs. 86.2%, p<0.01). TSAb was detected in all 17 M22-seronegative patients with untreated GD. In addition, in 8 of 10 M22- seronegative GD patients M22-TRAb became positive for 6-month of follow-up.

Conclusion: This study demonstrates that a newly modified TSAb assay is more useful in the differential diagnosis of GD from PT than conventional TSAb assay and highlights that a modified TSAb assay could demonstrate the existence of autoantibodies against TSHR in M22^TRAb-seronegative GD patients. Furthermore, in 80% of M22-seronegative GD patients, M22-TRAb appeared during follow-up.

Introduction

Graves’ disease (GD) is an autoimmune thyroid diseases caused by autoantibodies directed to the thyroid-stimulating hormone receptor (TSHR) [1-6]. The detection of autoantibodies against the TSHR (TRAb) has been clinically used as an aid in the differential diagnosis of hyperthyroidism or in monitoring of GD patients during treatment with antithyroid drug [7]. Until recently, TRAb measurements were performed using competitive inhibition of labeled TSH binding to the TSH receptor [6,8]. TRAb assays have improved over the years and finally as a third generation, a rapid and fully automated electrochemiluminescence immunoassay for TRAb called M22-TRAb assay which was used M22, a labeled thyroid-stimulating human monoclonal TSHR autoantibody [9], was developed. TRAb was detected by their ability to competitively inhibit M22-binding to solubilized porcine TSHR [10-13]. On the other hand, it has been reported that thyroid stimulating Ab (TSAb) , which is measured with conventional TSAb assay using porcine cells, was detected in only 89.3 % of GD patients and further 13 % of patients with Painless Thyroiditis (PT), thus making the differential diagnosis between GD and PT difficult [14]. In the present study, after a newly modified bioassay named TSAb kit ⌈Yamasa⌋ EIA (referred as TSAb) was developed, we report what are the modifications of the new assay that might improve the analytical performance at first. Next, we compared the analytical performance and clinical utility between conventional bioassay and TSAb assay that employs porcine cells. Furthermore, TSAb were measured using a modified TSAb assay in 17 M22-seronegative patients with untreated GD to investigate whether these cases have autoantibodies against the TSH receptor.

Materials and Methods

Subjects

Thirty-nine (3.6%) of 1.085 consecutive untreated GD patients were negative for M22-TRAb. Of them, 17 M22-TRAb-seronegative patients with untreated GD (39±17 years (M±SD); 1 man and 16 women) were included in this study.

In addition, 109 patients with newly diagnosed GD (39±13 years; 15 men and 94 women) and 94 patients with painless thyroiditis (PT) (35±12 years; 5 men and 89 women) were also selected for comparison between conventional TSAb assay and newly modified TSAb assay.

The diagnosis of GD was made on the basis of clinical and laboratory evidence of hyperthyroidism, the presence of a diffusely enlarged thyroid gland and increased diffuse thyroid uptake 20 minutes after Tc-99m injection (>2.0%) and/or increased vascularity index (>90%) in power color Doppler ultrasonography, as previously reported [8]. Inclusion criteria for M22-TRAb-seronegative Graves’ disease were undetected M22-TRAb (≤2.0IU/L) in addition to diagnostic standard of GD described above. The diagnosis of PT was based on laboratory evidence of thyrotoxicosis and decreased diffuse thyroid uptake of 20 minutes after Tc-99m injection (<0.5%) and/ or decreased vascularity (<50%) by power Doppler ultrasonography with negative M22-TRAb, as previously reported [8]. Finally, PT diagnosis is confirmed by spontaneous normalization of thyroid function during follow-up of a few months. Included also in this study were 110 normal controls (34±14 years; 17 men and 93 women) who were euthyroid with negative thyroid autoantibody and had normal thyroid volumes measured by thyroid ultrasonography. This study was approved by the Committee for Medical Research Ethics of the Kamijo Thyroid Research Institute and the Kamijo Thyroid Clinic, and all participants signed an informed consent form.

TSAb in patient sera was measured with TSAb kit ?Yamasa? EIA bioassay according to the manufacturer’s instruction (Yamasa Corporation, Chiba, Japan). This bioassay is specific to stimulating TSAb activation due to wild type receptor expressed on cultured porcine thyroid cells. Binding of TSAb in the patient to TSHR on porcine thyroid cells leads to activation of adenylate cyclase by interaction with the regulatory guanidine nucleotide binding (G) protein (Figure 1). Subsequently, detachment of the a-subunits from βγ complex activates adenylate cyclase through the effector to increase intracellular cAMP and release into culture medium.