MONOCLONAL-ANTIBODIES TO THE HUMAN TSH RECEPTOR - EPITOPE MAPPING ANDBINDING TO THE NATIVE RECEPTOR ON THE BASOLATERAL PLASMA-MEMBRANE OF THYROID FOLLICULAR CELLS

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target a utoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transfe rase or in a baculovirus-insect cell system, as a non-fusion glycoprot ein. To increase the epitope specificity of the mAbs, two different st rains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recogni zed by the mAbs were characterized by immunoblotting with various reco mbinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized e pitopes localized to different regions on the TSH-R.E; amino acids 22- 35 (A10 and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recogn ized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, sugge sting that this antibody reacts with the native receptor on thyroid ce lls. The binding of the mAbs A7, A10 and A11 was also restricted to th e basal surface of thyroid cells, but only after acetone fixation of t he sections, implying that the epitopes recognized on the amino and ca rboxyl terminus of the extracellular region of the receptor are not ac cessible on the native molecule. None of the mAbs stimulated cyclic AM P responses in COS-7 cells transiently transfected with full-length fu nctioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mA b A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the centr al region of the TSH-R.E without perturbing receptor function. The ava ilability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of t he autoantigenic regions on TSH-R implicated in disease activity.