Madl J E, Beitz A J, Johnson R L, Larson A A
J Neurosci. 1987 Sep;7(9):2639-50. doi: 10.1523/JNEUROSCI.07-09-02639.1987.
Aspartate is a putative excitatory amino acid neurotransmitter that is widely distributed in the CNS. To study its distribution, monoclonal antibodies were produced against beta-L-aspartyl-L-aspartate (beta-Asp-Asp) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde-borohydride. Three monoclonal antibodies, Asp1-3, were obtained with high degrees of selectivity for aldehyde-fixed aspartate. The immunocytochemical staining pattern of rat CNS was found to be similar for all 3 antibodies but differed in some regions from staining patterns produced by Glu1, a monoclonal antibody with high selectivity for a form of amide-linked glutamate. Tissue staining produced by Asp1-3 could be inhibited using aspartate conjugated to carrier proteins. Staining by Asp1 and Asp2 was also inhibited by free small molecules containing aspartate. Specificity of the 3 antibodies was evaluated by enzyme-linked immunoassay (ELISA) as follows: (1) reactivity of antibodies for conjugates of small molecules coated on ELISA plates; (2) ability of free small molecules to inhibit reactivity of antibodies for beta-Asp-Asp/KLH coated on ELISA plates; and (3) ability of conjugates to inhibit reactivity of antibodies for beta-Asp-Asp/KLH coated on ELISA plates. In all 3 types of assays, Asp1 and Asp2 displayed strong reactivity for small molecules and conjugates containing aspartate and little reactivity for small molecules and conjugates containing glutamate or GABA. Asp3 was highly reactive with conjugates containing aspartate using both directed and inhibition ELISA assays. For all 3 antibodies the precise staining pattern varied with the fixative used. Following glutaraldehyde fixation, dense immunocytochemical staining was observed in cerebral cortical neurons, some cerebellar granule cells, hippocampal pyramidal cells, and neurons of the inferior olivary nucleus. In addition, some putative GABAergic neurons, e.g., cerebellar basket and stellate cells, appeared to be stained. In general, acrolein fixation resulted in a more selective staining pattern in the CNS. For example, putative GABAergic neurons were no longer immunoreactive nor were hippocampal pyramidal cells.
天冬氨酸是一种假定的兴奋性氨基酸神经递质，广泛分布于中枢神经系统。为研究其分布情况，使用戊二醛 - 硼氢化钠将β - L - 天冬氨酰 - L - 天冬氨酸（β - Asp - Asp）与钥孔戚血蓝蛋白（KLH）偶联，以此制备单克隆抗体。获得了三种单克隆抗体，即Asp1 - 3，它们对醛固定的天冬氨酸具有高度选择性。发现大鼠中枢神经系统的免疫细胞化学染色模式对所有三种抗体而言是相似的，但在某些区域与Glu1产生的染色模式不同，Glu1是一种对酰胺连接形式的谷氨酸具有高选择性的单克隆抗体。Asp1 - 3产生的组织染色可用与载体蛋白偶联的天冬氨酸抑制。Asp1和Asp2的染色也可被含天冬氨酸的游离小分子抑制。通过酶联免疫吸附测定（ELISA）评估这三种抗体的特异性如下：（1）抗体对包被在ELISA板上的小分子偶联物的反应性；（2）游离小分子抑制抗体对包被在ELISA板上的β - Asp - Asp / KLH反应性的能力；（3）偶联物抑制抗体对包被在ELISA板上的β - Asp - Asp / KLH反应性的能力。在所有三种类型的测定中，Asp1和Asp2对含天冬氨酸的小分子和偶联物显示出强反应性，而对含谷氨酸或γ - 氨基丁酸的小分子和偶联物显示出弱反应性。使用直接ELISA和抑制ELISA测定，Asp3对含天冬氨酸的偶联物具有高反应性。对于所有三种抗体，精确的染色模式随所用固定剂而变化。经戊二醛固定后，在大脑皮质神经元、一些小脑颗粒细胞、海马锥体细胞和下橄榄核神经元中观察到密集的免疫细胞化学染色。此外，一些假定的γ - 氨基丁酸能神经元，如小脑篮状细胞和星状细胞，似乎也被染色。一般来说，丙烯醛固定导致中枢神经系统中更具选择性的染色模式。例如，假定的γ - 氨基丁酸能神经元和海马锥体细胞不再具有免疫反应性。
