PRODUCTION OF SPECIFIC ANTISERA AND MONOCLONAL-ANTIBODIES TO CHOLINE-ACETYLTRANSFERASE - CHARACTERIZATION AND USE FOR IDENTIFICATION OF CHOLINERGIC NEURONS
PRODUCTION OF SPECIFIC ANTISERA AND MONOCLONAL-ANTIBODIES TO CHOLINE-ACETYLTRANSFERASE - CHARACTERIZATION AND USE FOR IDENTIFICATION OF CHOLINERGIC NEURONS
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DOI：
10.1002/j.1460-2075.1982.tb01175.x
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发表时间：
1982-01-01
期刊：
影响因子：
11.4
通讯作者：
THOENEN, H
中科院分区：
文献类型：
--
作者：
ECKENSTEIN, F;THOENEN, H
从猪脑中纯化胆碱乙酰转移酶(ChAT)至> 95%同质性(纯化因子：646,000，纯化酶的比活性：形成128μmol乙酰胆碱/分钟/mg)。在十二烷基硫酸钠和β-巯基乙醇存在下，纯化的酶的凝胶电泳揭示了68,000道尔顿的单一蛋白质条带。免疫沉淀和双重免疫扩散测试表明，针对该蛋白的抗血清可特异性识别 ChAT。针对该酶制备的单克隆抗体特异性结合来自粗制猪脑上清液的蛋白质，其MW为68,000，比活性为153μmol/min/mg。该抗体没有表现出物种交叉反应性。通过比较使用抗血清和使用单克隆抗体获得的猪视网膜标记，确定了 ChAT 免疫组织化学定位的特异性。两种探针特异性识别相同的视网膜结构：在内核层和神经节细胞层中发现标记的细胞体，而在内丛状层中染色双条带。在大鼠脊髓中，抗血清标记位于中间外侧核、闰区和中央自主神经区的运动神经元和节前交感神经元。
Choline acetyltransferase (ChAT) was purified from pig brain to > 95% homogeneity (purification factor: 646,000, specific activity of the purified enzyme: 128 .mu.mol acetylcholine formed/min per mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate and .beta.-mercaptoethanol revealed a single protein band at 68,000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a MW of 68,000 and a specific activity of 153 .mu.mol/min per mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT was established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermediolateral nucleus, the intercalated region and the central autonomic area.