BINDING CHARACTERISTICS OF ANTIBODIES TO THE TSH RECEPTOR

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polycl onal antibodies and a panel (n=5) of monoclonal antibodies to the extr acellular fragment of the TSHR. The binding characteristics of the ant ibodies to linear, conformational, glycosylated and unglycosylated for ms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Weste rn blotting with both native and recombinant human TSHR expressed in C HO cells, immunoprecipitation of S-35-labelled full-length TSHR produc ed in an in vitro transcription/ translation system, immunoprecipitati on of I-125- TSH/TSHR complexes, inhibition of I-125-TSH binding to th e TSHR and fluorescence activated cell sorter (FACS) analysis of bindi ng to CHO-K1 cells expressing the TSHR on their cell surface. Fab frag ments of monoclonal antibodies were isolated, labelled with I-125 and used to determine the affinity constants of these antibodies with rece ptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies react ed with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit I-125-TSH binding to native human TSHR (74% inhib ition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7)M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with T SHR expressed on the surface of CHO-K1 cells. The FACS unreactive mono clonal (3C7) bound well to detergent solubilised TSH receptors and thi s emphasised the importance of using a combination of FACS analysis an d radioactively-labelled probes in analysis of the TSH receptor. The m onoclonal antibodies produced in this study were found to be of relati vely ion: affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.