Measurement of Thyroid-stimulating Hormone Receptor Autoantibodies by ELISA

Abstract

Hyperthyroidism in Graves disease is attributable to autoantibodies to the thyroid-stimulating hormone receptor (TSHR), and measurement of these TSHR autoantibodies (TRAbs) can be useful in disease diagnosis and management (1)(2)(3)(4). Usually TRAbs are detected by bioassays based on cultured cells or by receptor assays based on 125I-labeled TSH (1)(2)(3)(4). The most widely used receptor assay (5) uses detergent-solubilized porcine TSHR with TRAbs to inhibit the TSHR-125I-TSH interaction, and polyethylene glycol (PEG) to separate receptor-bound and free labeled TSH by precipitation. Recently, we developed a monoclonal antibody (MAb) to the porcine TSHR COOH terminus that can be used to attach the TSHR to a solid phase, and we describe the use of this antibody to develop an ELISA for TRAbs. In the assay, TRAbs compete for binding to the TSHR with biotinylated bovine TSH [bovine TSH being readily available and having a much higher biological activity than human TSH (6)]. TSH-biotin binding is then monitored using a streptavidin-peroxidase conjugate. The C-terminal end of the porcine TSHR (last 160 amino acids) (3) was expressed in Escherichia coli as a fusion protein with glutathione S -transferase and used to produce MAbs as described previously (7). One of the MAbs had a relatively high affinity (5 × 109 L/mol) for detergent-solubilized porcine TSHR, and the purified IgG was used to coat ELISA plate wells by incubation overnight at 4 °C. In these experiments, 100 μL of 0.01 g/L antibody in 100 mmol/L NaHCO3, pH 9.2, was used. After washing and coating with 10 g/L bovine …