Detection of human and rodent 5-HT(3B )receptor subunits by anti-peptide polyclonal antibodies

BACKGROUND: The 5-HT(3 )receptor is a member of a neurotransmitter-gated ion channel family which includes nicotinic acetylcholine, GABA(A), and glycine receptors. While antibodies specific for the 5-HT(3A )receptor subunit are plentiful, and have revealed a wealth of structural and functional information, few antisera exist for the detection of 5-HT(3B )receptor subunits. Here we describe the generation and characterisation of a rabbit polyclonal antiserum that specifically recognises 5-HT(3B )receptor subunits RESULTS: Immunization of a rabbit with a 20-mer peptide, corresponding to the N-terminus of the human 5-HT(3B )receptor subunit, generated serum with polyclonal antibodies from which an IgG fraction was purified, yielding pAb77. The antibodies were shown to label 5-HT(3B )receptor subunits in transfected human embryonic kidney cells and rodent tissues using Western blots. Immunocytochemistry using pAb77 on these cells showed that 5-HT(3B )receptor subunits do not reach the plasma membrane in the absence of 5-HT(3A )receptor subunits. Immunohistochemical analysis of rat brain sections showed pAb77 immunoreactivity in distinct populations of cells in the hippocampus. CONCLUSION: We have demonstrated that pAb77 antibodies specifically label native and recombinant 5-HT(3B )receptor subunits with high affinity and specificity. The antibody was shown to be useful for the determination of both receptor trafficking and also mapping 5-HT(3B )receptor subunit expression in the CNS.