Autoantibodies against the thyroid-stimulating hormone receptor (TSHR) are associated with autoimmune thyroid diseases. These TSHR autoantibodies (TRAb) bind to TSHR and are categorized into two types based on their biological activity: thyroid-stimulating antibody (TSAb) and thyroid-blocking antibody (TBAb). TSAbs have been implicated in the pathogenesis of Graves' disease (Smith and Hall, 1974). TBAbs are detected in some patients with hypothyroidism.

TRAb levels can be quantified using an automated competitive immunoassay with the Ruthenium-labeled TSHR-stimulating monoclonal antibody M22 (Tozzoli et al., 2010). Although these binding assays are rapid (approximately 30 min), they cannot differentiate between TSAb and TBAb. TSAb levels can be measured based on the cAMP production activity via TSHR stimulation of the antibody against cells expressing TSHR, and it is possible to evaluate the stimulating activity of TSHR, which cannot be evaluated by measuring TRAb concentration. There are two conventional methodologies for TSAb measurement: One method uses a bioassay based on a cyclic adenosine monophosphate (cAMP)-dependent reporter gene assay, which requires a considerable amount of time (18–21 h) for analysis (Lytton et al., 2010). The other approach combines a porcine primary thyroid cell bioassay with cAMP detection in a TSAb enzyme immunoassay (TSAb-EIA; Kamijo and Togashi, 2014). The TSAb-EIA method enhances the diagnostic predictive value by amplifying the positivity rate of untreated Graves' disease and diminishing the positivity rate of silent thyroiditis compared to previous radio-immunoassays (Kamijo and Togashi, 2014); however, TSAb-EIA techniques have several technical drawbacks. The process involves numerous operational steps, including a 4 h serum-cell reaction, cell lysis, and immunoassay, and overall takes approximately 6 h. Another reported TSAb measurement technique, the aequorin-TSAb assay, involves live-cell TSAb bioassays using cAMP-dependent calcium channels and aequorin, but also requires 4 h for analysis (Araki et al., 2015). Their multistep nature and protracted processing times necessitate substantial simplification and acceleration.

The recent advent of luciferase-based cAMP biosensors has enabled real-time monitoring of increased intracellular cAMP concentrations (Binkowski et al., 2009; Buccioni et al., 2011; Kumar et al., 2017). Leveraging this innovation, we developed a novel, next-generation rapid TSAb measurement method (rapid-TSAb assay) employing a luciferase-based cAMP biosensor. This assay can assess TSAb activity within an approximate timeframe of only 1 h.

TSHR constructs used for the different systems vary; TSAb-EIA employs porcine wild-type TSHR, the aequorin-TSAb assay uses human wild-type TSHR (wt-TSHR), and the reporter gene assay utilizes chimeric TSHR (Mc4) (Araki et al., 2015; Kamijo and Togashi, 2014; Tozzoli et al., 2010). The Mc4 construct contains amino acids 261–370 within the TSHR N-terminal extracellular domain replaced by sequences from the rat luteinizing hormone/chorionic gonadotropin hormone receptor. Recently, a TSAb bioassay based on the same principle, using a CHO-K1 cell line expressing a cAMP biosensor and Mc4 has been previously reported (Miao et al., 2022). However, the effects of differences in receptors and the measurement principles on stimulatory activity remain unclear.

In this study, we describe the performance of a novel, rapid-TSAb assay using human wt-TSHR. We simultaneously performed a comparison to assess cAMP stimulatory activity between the wt-TSHR and Mc4 constructs.