Hill B L, Erlanger B F
Department of Microbiology, Columbia University, New York, New York 10032.
Endocrinology. 1988 Jun;122(6):2840-50. doi: 10.1210/endo-122-6-2840.
Monoclonal antibodies that bind to the TSH receptor were obtained by an autoantiidiotypic approach in which immunization of BALB/c mice was performed with mixtures of bovine (b) and human (h) TSH. Two of 28 positive wells were selected for cloning and characterization: D2 and 4G11. Their antiidiotypic character was evidenced by TSH-inhibitable binding to affinity-purified polyclonal anti-TSH. The specificity of D2 and 4G11 for the hormone-binding region of the TSH receptor was demonstrated by several findings: 1) they inhibited the binding of [125I]iodo-bTSH to receptor in a dose-dependent manner; 2) their binding to partially purified thyroid plasma membranes could be completely inhibited by bTSH and hTSH; and 3) they inhibited the TSH-dependent growth and adenylate cyclase stimulation in FRTL-5 cells in a dose-dependent manner. By Western blot analysis of bovine thyroid membranes, D2 bound to a polypeptide of 188,000-195,000 mol wt under nonreducing conditions and 54,000-59,000 mol wt after treatment of membranes with beta-mercaptoethanol; the 4G11 epitope was undetectable. Scatchard analysis of the binding of 125I-labeled antibodies to receptor showed that 4G11 bound to a single site with a Kd of 5.7 X 10(-9) M, whereas D2 showed complex binding characterized by high affinity (Kd = 1.74 X 10(-11) M) and low affinity (Kd = 1.3 X 10(-8) M) sites. Binding studies in which D2 and 4G11 competed with each other for the TSH receptor showed mutual but unequal inhibition. The data suggest that portions of the D2 and 4G11 epitopes overlap, but that there is a high affinity binding site(s) for D2 for which 4G11 competes less effectively. The binding of D2 and 4G11 to TSH receptor was inhibited by monoclonal antibodies secreted by Graves' heterohybridomas, showing that D2 and 4G11 share characteristics with autoantibodies of Graves' disease and lending support to the hypothesis that idiotypic network interactions may play a role in the pathogenesis of Graves' disease.
通过自身抗独特型方法获得了与促甲状腺激素（TSH）受体结合的单克隆抗体，该方法是用牛（b）和人（h）TSH的混合物对BALB/c小鼠进行免疫。从28个阳性孔中选择了两个进行克隆和鉴定：D2和4G11。它们的抗独特型特性通过与亲和纯化的多克隆抗TSH的TSH抑制性结合得到证实。D2和4G11对TSH受体激素结合区域的特异性通过以下几个发现得以证明：1）它们以剂量依赖性方式抑制[125I]碘 - bTSH与受体的结合；2）bTSH和hTSH可完全抑制它们与部分纯化的甲状腺质膜的结合；3）它们以剂量依赖性方式抑制FRTL - 5细胞中TSH依赖性生长和腺苷酸环化酶的刺激。通过对牛甲状腺膜的蛋白质印迹分析，在非还原条件下D2与分子量为188,000 - 195,000道尔顿的多肽结合，在用β - 巯基乙醇处理膜后与分子量为54,000 - 59,000道尔顿的多肽结合；4G11的表位无法检测到。对125I标记抗体与受体结合的Scatchard分析表明，4G11以Kd为5.7×10（-9）M的单一结合位点结合，而D2表现出以高亲和力（Kd = 1.74×10（-11）M）和低亲和力（Kd = 1.3×10（-8）M）位点为特征的复杂结合。D2和4G11相互竞争TSH受体的结合研究显示出相互但不等效的抑制作用。数据表明D2和4G11表位的部分重叠，但存在D2的高亲和力结合位点，4G11对其竞争效果较差。D2和4G11与TSH受体的结合被格雷夫斯病异源杂交瘤分泌的单克隆抗体抑制，表明D2和4G11与格雷夫斯病自身抗体具有共同特征，并支持独特型网络相互作用可能在格雷夫斯病发病机制中起作用的假说。
