Simmons M L, Frondoza C G, Coyle J T
Department of Neuroscience, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.
Neuroscience. 1991;45(1):37-45. doi: 10.1016/0306-4522(91)90101-s.
N-Acetyl-aspartate is found in high concentrations in all areas of the brain, but is undetectable in non-neuronal tissue. In order to characterize the cellular localization of N-acetyl-aspartate in brain, highly specific monoclonal antibodies against N-acetyl-aspartate were produced by fusing spleen lymphocytes obtained from mice immunized with N-acetyl-aspartate conjugated to thyroglobulin by carbodiimide with P3/x63-Ag8.653 mouse myeloma cells. Clones were selected which secrete IgG2a(k) antibodies highly specific for conjugated N-acetyl-aspartate. Only 3-6% cross-reactivity with conjugated N-acetyl-aspartate-glutamate was observed at high antibody concentrations, whereas no cross-reactivity (less than 1%) was observed with conjugated N-acetyl-glutamate or aspartate. Preincubation of the antibodies with 0.5 mg/ml conjugated N-acetyl-aspartate blocked immunoreactivity more than 90%, while preincubation with conjugated N-acetyl-aspartate-glutamate and free N-acetyl-aspartate had no effect. Immunocytochemical staining has shown that N-acetyl-aspartate-like immunoreactivity is localized in neurons, which are widely distributed throughout the brain. The immunoreactive neurons exhibited intense staining of the perikarya, proximal dendrites and axons. No consistent pattern of distribution of immunoreactivity was observed with regard to primary neurotransmitter characteristics of stained neurons although neurons with long projections or extensive arbors, such as pyramidal cells in cortex, locus coeruleus, motor neurons and Purkinje cells, stained much more intensively than local circuit neurons.
N-乙酰天门冬氨酸在大脑所有区域均以高浓度存在，但在非神经组织中无法检测到。为了确定大脑中N-乙酰天门冬氨酸的细胞定位，通过将用碳二亚胺与甲状腺球蛋白偶联的N-乙酰天门冬氨酸免疫的小鼠脾脏淋巴细胞与P3/x63-Ag8.653小鼠骨髓瘤细胞融合，制备了针对N-乙酰天门冬氨酸的高度特异性单克隆抗体。筛选出分泌对偶联的N-乙酰天门冬氨酸具有高度特异性的IgG2a(k)抗体的克隆。在高抗体浓度下，仅观察到与偶联的N-乙酰天门冬氨酸-谷氨酸有3-6%的交叉反应性，而与偶联的N-乙酰谷氨酸或天门冬氨酸未观察到交叉反应性（小于1%）。抗体与0.5mg/ml偶联的N-乙酰天门冬氨酸预孵育可使免疫反应性阻断超过90%，而与偶联的N-乙酰天门冬氨酸-谷氨酸和游离N-乙酰天门冬氨酸预孵育则无影响。免疫细胞化学染色表明，N-乙酰天门冬氨酸样免疫反应性定位于广泛分布于整个大脑的神经元中。免疫反应性神经元的胞体、近端树突和轴突呈现强烈染色。尽管具有长投射或广泛分支的神经元，如皮质锥体细胞、蓝斑、运动神经元和浦肯野细胞，染色比局部回路神经元强烈得多，但在染色神经元的主要神经递质特征方面未观察到一致的免疫反应性分布模式。
