De Blas A L, Sangameswaran L
Life Sci. 1986 Nov 24;39(21):1927-36. doi: 10.1016/0024-3205(86)90316-4.
Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.
用苯二氮䓬 - 牛血清白蛋白偶联物免疫BALB/c小鼠后，产生了4株分泌抗苯二氮䓬单克隆抗体的杂交瘤细胞系。从腹水液中纯化单克隆抗体，并测定它们对苯二氮䓬及其他苯二氮䓬受体配体的结合亲和力。这些抗体对地西泮、氟硝西泮、Ro5 - 4864、Ro5 - 3453、Ro11 - 6896和Ro5 - 3438具有非常高的结合亲和力（解离常数Kd值在10⁻⁹ M范围内）。然而，这些抗体对苯二氮䓬受体反向激动剂（β - 咔啉）和拮抗剂（Ro15 - 1788和CGS - 8216）的亲和力非常低。其中一种单克隆抗体（21 - 7F9）已被用于证明脑中存在苯二氮䓬样分子，并用于纯化这些分子。免疫细胞化学实验表明，这些分子是神经元性的而非胶质细胞性的，并且它们在脑中广泛分布。免疫印迹表明几种脑肽中存在苯二氮䓬样表位。一种与中枢型苯二氮䓬受体结合且具有激动剂特性的内源性物质已从牛脑中纯化至同质。纯化过程包括在固定化单克隆抗苯二氮䓬抗体上进行免疫亲和层析，随后在Sephadex G - 25上进行凝胶过滤以及两次反相高效液相色谱。纯化后的物质分子量较小，其活性对蛋白酶具有抗性。该内源性物质可阻断激动剂、反向激动剂和拮抗剂与中枢型苯二氮䓬受体的结合，但不抑制Ro5 - 4864与外周型苯二氮䓬受体的结合。神经递质γ - 氨基丁酸可增加苯二氮䓬受体对纯化物质的亲和力。初步证据表明，纯化后的物质是一种苯二氮䓬，其分子结构与N - 去甲基地西泮相同或非常接近。
