Characterization of an antiserum to glycyl-d-aspartate (GDA) and its use as a probe for endogenous...

The long-term goal of this study is to identify endogenous N-methyl-d-aspartate (NMDA)-like compounds with the help of antibodies. Since NMDA contains a blocked amino group, it cannot be conjugated with glutaraldehyde to a carrier protein for immunization. Thus, we used the synthetic dipeptide GDA as a model hapten. The resulting antisera were characterized in both immunocytochemistry and enzyme immunoassay. A limited survey of immuno-cytochemically stained paraffin and vibratome sections revealed positively stained neurons in several areas of the central nervous system, e.g., the cerebral cortex, the hippocampus, and the trigeminal and spinal ganglia. The specificity of the staining was determined on paraffin sections of trigeminal ganglion. GDA, NMDA, and d-aspartate blocked staining, whereas l-aspartate, l-glutamate, kainic acid, and quisqualic acid had little or no effect. An enzyme immunoassay was developed in which polystyrene plates were coated with 10 muM GDA and A-GDA 641 used at a 1:150,000 dilution. In competition studies GDA had an EC(50) = 8.8 x 10(-7)M and a detection limit of 2.3 x 10(-7)M. Of the 25 other compounds tested only succinic acid, d-aspartate, quinolinic acid, NMDA, and fumaric acid demonstrated significant cross-reactivity (>0.01%) with the antiserum. In addition extracts of both brain and liver displaced the binding of the antiserum to the GDA-coated plate, although there was no immunocytochemical labeling in liver tissue. The results show that the GDA serum has strict and specific binding requirements for a d-aspartate-like chemical configuration and recognizes endogenous brain substance(s). The identity and function of that substance (or substances) remain to be determined.