Production and characterization of a specific 5-HT₂ receptor antibody

A synthetic peptide was used to generate antibodies against the rat serotonin-2 (5-HT₂) receptor. The peptide corresponds to a unique sequence from the N-terminal extracellular portion of the receptor protein (antibody = Ab 5HT2-N). This peptide was chosen based on its theoretical antigenic index and for specificity to the 5-HT₂ receptor. In dot blot analysis, antisera detected 2 ng-2 μg of synthetic peptide at dilutions of 1/200-1/20,000. COS-7 cells transiently transfected with a eularyotic expression vector containing the 5-HT₂ cDNA displayed intense immunoreactivity with crude and affinity-purified Ab 5HT2-N. In contrast, no immunoreactivity was seen in control experiments when: (1) non-transfected or vector transfected COS-7 cells were used; (2) pre-immune sera was substituted for primary antisera; (3) primary antisera was omitted; or (4) antiserum was pre-adsorbed to 10 μM synthetic peptide. Immunohistochemical analysis of sections of perfused rat brain revealed intense immunolabelling of a subset of neurons in regions of the ventral forebrain, dorsal hippocampus, striatum, cerebral cortex, and laterodorsal tegmental nucleus (LDT). An especially dense band of small cells was seen in layer 2 of pyriform cortex. There was a very high concentration of labelled cells in the laterodorsal tegmental nucleus. In situ hybridization histochemistry with a 5-HT₂ antisense cRNA riboprobe showed a pattern of hybridization in forebrain similar to the pattern of immunolabelling with Ab 5HT2-N. Western blot analysis of proteins extracted from the LDT revealed a single protein species reacting with the antibody. This reactivity is not present in the pre-immune sera and is blocked by the synthetic antigen. These data suggest that we have produced a specific antibody suitable for immunocytochemical application that recognizes an N-terminal epitope of the rat 5-HT₂ receptor.