Cayanis E, Rajagopalan R, Cleveland W L, Edelman I S, Erlanger B F
J Biol Chem. 1986 Apr 15;261(11):5094-103.
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.
通过自身抗独特型途径，使用与甲状腺球蛋白偶联的曲安西龙衍生物免疫小鼠，获得了一种针对糖皮质激素受体配体结合位点附近区域的单克隆抗体（8G11 - C6）。用亲和纯化的多克隆兔抗曲安西龙抗体的Fab片段筛选所得杂交瘤，以寻找抗独特型抗体（抗抗类固醇抗体）。然后通过去除法筛选抗独特型抗体与大鼠细胞质糖皮质激素受体的结合情况，得到一个克隆8G11 - C6，其对受体的特异性通过蔗糖密度分析和蛋白质印迹分析得到验证。用[3H]曲安奈德丙酮化物预标记细胞质后，去除作用并未显著降低。抗独特型抗体（8G11 - C6）以浓度依赖的方式与抗类固醇的Fab片段和部分纯化的受体结合。只有已知与糖皮质激素受体结合的类固醇的兔血清白蛋白缀合物能抑制这两种结合反应。赖氨酸和含多达六个氨基酸的寡肽的曲安西龙衍生物抑制抗独特型抗体与Fab片段的结合，但不抑制与受体的结合，这意味着抗类固醇抗体的靶表位可能比受体的交叉反应表位更靠近其糖皮质激素结合位点。我们的研究结果进一步证明了自身抗独特型途径在制备抗受体抗体方面的多功能性。
