Evaluation of the immunocytochemical method for amino acids

Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.