Monoclonal antibodies specific for fixative-modified aspartate: immunocytochemical localization in the rat CNS

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.