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Identification of Serotonin Receptors Recognized by Anti‐Idiotypic Antibodies
Author(s) -
Tamir Hadassah,
Liu Kuopeing,
Hsiung Shuchi,
Yu Peiying,
Kirchgessner Annette L.,
Gershon Michael D.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb08240.x
Subject(s) - antibody , affinity chromatography , microbiology and biotechnology , polyclonal antibodies , antiserum , receptor , chemistry , 5 ht receptor , biochemistry , biology , ligand (biochemistry) , serotonin , immunology , enzyme
Anti‐idiotypic antibodies were generated by immunizing rabbits with affinity‐purified antibodies to serotonin (5‐hydroxytryptamine; 5‐HT). Anti‐5‐HT activity was removed from the resulting antisera by chromatography through a 5‐HT affinity column. The anti‐idiotypic antibodies were demonstrated by enzyme‐linked immunosorbent assay to bind to affinity‐purified whole anti‐5‐HT antibodies and their Fab fragments. Anti‐idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5‐HT were coupled, competed with 5‐HT (covalently bound to protein) for the binding sites on anti‐5‐HT antibodies and serotonin binding protein. The anti‐idiotypic antibodies antagonized the binding of [ 3 H]5‐HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti‐ idiotypic antibodies also blocked the binding of the 5‐HT 1B ‐ selective ligand (‐)‐[ 125 I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti‐idiotypic antibodies failed to inhibit binding of the 5‐ HT 1A ‐selective ligand 8‐hydroxy‐2‐(di‐ n )‐[ 3 H]propylamino)‐ tetralin ([ 3 H]8‐OH‐DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti‐idiotypic antibodies may recognize some 5‐HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti‐idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5‐ HT 1C or 5‐HT 2 receptor or with a genomic clone encoding the 5‐HT 1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5‐ HT 1C and 5‐HT 2 receptors, but not on the surfaces of HeLa cells that expressed 5‐HT 1A receptors. Immunostaining of cells by the anti‐idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5‐HT 1C receptors was blocked by mesulergine (but not ketanserin, 8‐OH‐DPAT. or spiperone), whereas that of cells expressing 5‐HT 2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8‐OH‐DPAT). The anti‐ idiotypic antibodies failed to inhibit the uptake of [ 3 H]5‐HT by serotonergic neurons. It is concluded that the anti‐idiotypic antibodies generated with anti‐5‐HT serum recognize the 5‐ HT 1B , 5‐HT lC , and 5‐HT 2 receptor subtypes; however, neither 5‐HT 1A receptors nor 5‐HT uptake sites appear to react with these antibodies.