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Notes: Abstract: Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 ± 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44–10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44–10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44–10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with Mab 44–10 were located immunocytochemically in sections of rat brain. 5-HT immunoreactivity was localized simultaneously in the same sections by using affinity-purified rabbit anti-5-HT antibodies and species-specific secondary antibodies coupled to a contrasting fluorophore. MAb 44–10 immunostaining involved neuronal cell bodies, neurites, and terminals. This immunostaining was intense within the nuclei of the median raphe and the B9 cell group. Coincident expression with 5-HT was observed; however, MAb 44–10 also immunostained many neurons in which 5-HT immunoreactivity was not seen. These observations may indicate that SBP is distributed more widely in the brain than 5-HT; however, because SBP immunoreactivity is not found in nonserotonergic neurons when monospecific polyclonal antibodies are used for immunocytochemistry, it seems more likely that some nonserotonergic neurons contain another protein (such as the 68-kDa SBP) that also contains an epitope recognized by MAb 44–10. Nevertheless, these data demonstrate that MAb 44–10 reacts with the 5-HT binding domain of 45-kDa and 56-kDa SBP and will be a valuable tool for analyzing these proteins.