Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor.
A monoclonal antibody (8G11-C6) that is directed to a region near the ligand-binding site of the glucocorticoid receptor was obtained by an auto-anti-idiotypic route, using a derivative of triamcinolone coupled to thyroglobulin to immunize a mouse. The resulting hybridomas were screened for anti-idiotypic antibody (anti-antisteroid) with Fab fragments of affinity-purified polyclonal rabbit anti-triamcinolone antibody. The anti-idiotypes were then screened for binding to rat cytosol glucocorticoid receptor by a depletion procedure, yielding a clone, 8G11-C6, whose specificity for receptor was verified by sucrose density and Western blot analyses. Depletion was not significantly reduced by prelabeling the cytosol with [3H]triamcinolone acetonide. The anti-idiotype (8G11-C6) bound to Fab fragments of antisteroid and to partially purified receptor in a concentration-dependent manner. Both binding reactions were inhibited only by rabbit serum albumin conjugates of steroids known to bind to the glucocorticoid receptors. Triamcinolone derivatives of lysine and of oligopeptides containing up to six amino acids inhibited the binding of the anti-idiotype to the Fab fragments but not to the receptor, implying that the target epitope of the antisteroid antibody may be closer to its glucocorticoid-binding site than the cross-reacting epitope of the receptor. Our findings demonstrate further the versatility of the auto-anti-idiotypic route for the preparation of anti-receptor antibodies.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001323:Autoantibodies / Q000096:biosynthesis*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D008099:Liver / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000276:immunology* / Q000378:metabolism; D013954:Thyroglobulin / Q000276:immunology; D014222:Triamcinolone Acetonide / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D007130:Immunoglobulin Idiotypes; D011965:Receptors, Glucocorticoid; D013954:Thyroglobulin; D014222:Triamcinolone Acetonide; C005900:triamcinolone hexacetonide