Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.