Detection of human and rodent 5-HT3B receptor subunits by anti-peptide polyclonal antibodies.
BACKGROUND
The 5-HT3 receptor is a member of a neurotransmitter-gated ion channel family which includes nicotinic acetylcholine, GABAA, and glycine receptors. While antibodies specific for the 5-HT3A receptor subunit are plentiful, and have revealed a wealth of structural and functional information, few antisera exist for the detection of 5-HT3B receptor subunits. Here we describe the generation and characterisation of a rabbit polyclonal antiserum that specifically recognises 5-HT3B receptor subunits


RESULTS
Immunization of a rabbit with a 20-mer peptide, corresponding to the N-terminus of the human 5-HT3B receptor subunit, generated serum with polyclonal antibodies from which an IgG fraction was purified, yielding pAb77. The antibodies were shown to label 5-HT3B receptor subunits in transfected human embryonic kidney cells and rodent tissues using Western blots. Immunocytochemistry using pAb77 on these cells showed that 5-HT3B receptor subunits do not reach the plasma membrane in the absence of 5-HT3A receptor subunits. Immunohistochemical analysis of rat brain sections showed pAb77 immunoreactivity in distinct populations of cells in the hippocampus.


CONCLUSIONS
We have demonstrated that pAb77 antibodies specifically label native and recombinant 5-HT3B 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-HT3B receptor subunit expression in the CNS.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. dreeves@aecom.yu.edu
David C
DC
Reeves
Sarah C R
SC
Lummis
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D007150:Immunohistochemistry; D010455:Peptides / Q000276:immunology; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D051381:Rats; D044406:Receptors, Serotonin, 5-HT3 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*
D007074:Immunoglobulin G; D010455:Peptides; D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3