Monoclonal Antibodies Against Glutaraldehyde‐Conjugated Dopamine

Four mice were immunized with dopamine (DA)—glutaraldehyde (G)—protein conjugates over a period of 8–10 weeks. Polyclonal antisera, obtained at various intervals, were tested using an enzyme‐linked immuno‐sorbent assay (ELISA). All had anti‐conjugated DA antibodies. As soon as good antibody affinity was detected between 10 ‐10 and 10 ‐6 M , the mouse yielding the highest apparent affinity was killed, and the spleen was dissected out. Hybridomas were obtained from spleen cells fused with SP 2 /O/Ag myeloma cells. Supernatant culture media of hybridomas were tested for the presence of anti‐conjugated DA antibodies with the ELISA method. Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique. The resulting supernatant culture media were again tested by ELISA. Clones that gave a high antibody affinity (10 ‐10 ‐10 ‐8 M ) for G‐conjugated DA were used for histochemical localization of DA in rat brain. G‐fixed rat brains were sectioned from the telencephalon to the mesencephalon, reduced with sodium borohydride, and prepared for peroxidase‐antiperoxidase immunocytochemistry using supernatant (diluted 1:100) or ascites fluid (diluted 1:50,000). Dense networks of very fine fibers were observed in the striatum, septum, and cortex. Numerous immunoreactive cell bodies were found in the ventral tegmental area, the substantia nigra, the hypothalamus, and the dorsal raphe. The ELISA tests and adsorption controls suggested that the monoclonal antibody allowed highly specific detection of DA in tissues.