A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT4) receptor.
Splenocytes from a BALB/c mouse immunised with a synthetic peptide corresponding to the second extracellular loop of the 5-HT4 receptor were fused with SP2/O myeloma cells to produce a monoclonal antibody. The monoclonal antibody was of the IgG2b isotype. The antibody recognised the human 5-HT4(g) (h5-HT4(g)) receptor by immunoblots and by immunofluorescence on chinese hamster ovary (CHO) cells expressing this 5-HT4 receptor isoform. Epitope mapping of the antibody suggested the recognition of a conformational epitope, encompassing the N- and C-terminal fragments of the second extracellular loop. Kinetic experiments using surface plasmon resonance showed that the antibody had a picomolar affinity for its cognate peptide. Inhibition experiments using the same methodology confirmed the specificity of the interaction. The antibody at a concentration of 500 pM competitively inhibited inverse agonist GR113808 binding and showed an inverse agonist effect on the basal activity of CHO cells expressing the 5-HT4(g) receptor. The antibody decreased the effect of 5-HT at 500 and 50 pM concentrations but it increased 5-HT-induced cAMP levels at 5 pM. The dual effect of the monoclonal antibody could be ascribed to mono- or bivalent recognition of the receptor. The antibody described here is the first example of a high-affinity modulator of the 5-HT4 receptor.
Kamel
Rehab
R
UPR 9021 du C.N.R.S., Immunologie et Chimie Thérapeutiques, I.B.M.C., 15, Rue René Descartes, F-67084 Strasbourg, France.
Eftekhari
Pierre
P
Garcia
Simone
S
Berthouze
Magali
M
Berque-Bestel
Isabelle
I
Peter
Jean-Christophe
JC
Lezoualc'h
Frank
F
Hoebeke
Johan
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D044422:Receptors, Serotonin, 5-HT4 / Q000276:immunology*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D044422:Receptors, Serotonin, 5-HT4