Generation of a mouse monoclonal TSH receptor antibody with stimulating activity

A Balb/c mouse was subjected to genetic immunization with a cDNA construct encoding the human thyrotropin receptor (TSHr). The immune response of the mouse resulted in the production of immunoglobulins recognizing the TSHr in three different assays: (1) flow immunocytometry (FACS) with CHO cells expressing the receptor; (2) receptor-dependent stimulation of cAMP production in the same cell line; and (3) competition with labeled TSH for binding to the receptor. One thousand hybridomas were generated from the spleen of the mouse and their supernatants were screened. A single monoclonal, IRI-SAb1, scored positive in all three assays and was studied further. It stimulated 13-fold cAMP production in TSHr-expressing CHO cells, with an EC50 in the low nanomolar range. When compared with bovine TSH, IRI-SAb1 behaved as a partial agonist. Contrary to the expectation from the characteristic of autoantibodies of Graves' patients, IRI-SAb1 recognized a linear epitope, which was localized in a segment encompassing the first 281 residues of the receptor.