Development and characterization of monoclonal antibodies specific to the serotonin 5-HT2A receptor.
Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.
Wu
C
C
Molecular and Cellular Biology, PharMingen, San Diego, California, USA.
Yoder
E J
EJ
Shih
J
J
Chen
K
K
Dias
P
P
Shi
L
L
Ji
X D
XD
Wei
J
J
Conner
J M
JM
Kumar
S
S
Ellisman
M H
MH
Singh
S K
SK
D000368:Aged; D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D017966:Pyramidal Cells / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012583:Schwann Cells / Q000378:metabolism
D000911:Antibodies, Monoclonal; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins