Production and characterization of a specific 5-HT2 receptor antibody.
A synthetic peptide was used to generate antibodies against the rat serotonin-2 (5-HT2) receptor. The peptide corresponds to a unique sequence from the N-terminal extracellular portion of the receptor protein (antibody = Ab 5HT2-N). This peptide was chosen based on its theoretical antigenic index and for specificity to the 5-HT2 receptor. In dot blot analysis, antisera detected 2 ng-2 micrograms of synthetic peptide at dilutions of 1/200-1/20,000. COS-7 cells transiently transfected with a eukaryotic expression vector containing the 5-HT2 cDNA displayed intense immunoreactivity with crude and affinity-purified Ab 5HT2-N. In contrast, no immunoreactivity was seen in control experiments when: (1) non-transfected or vector transfected COS-7 cells were used; (2) pre-immune sera was substituted for primary antisera; (3) primary antisera was omitted; or (4) antiserum was pre-adsorbed to 10 microM synthetic peptide. Immunohistochemical analysis of sections of perfused rat brain revealed intense immunolabelling of a subset of neurons in regions of the ventral forebrain, dorsal hippocampus, striatum, cerebral cortex, and laterodorsal tegmental nucleus (LDT). An especially dense band of small cells was seen in layer 2 of pyriform cortex. There was a very high concentration of labelled cells in the laterodorsal tegmental nucleus. In situ hybridization histochemistry with a 5-HT2 antisense cRNA riboprobe showed a pattern of hybridization in forebrain similar to the pattern of immunolabelling with Ab 5HT2-N. Western blot analysis of proteins extracted from the LDT revealed a single protein species reacting with the antibody. This reactivity is not present in the pre-immune sera and is blocked by the synthetic antigen.(ABSTRACT TRUNCATED AT 250 WORDS)
Garlow
S J
SJ
Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Department of Psychiatry, Stanford University Medical School, CA 94305-5485.
Morilak
D A
DA
Dean
R R
RR
Roth
B L
BL
Ciaranello
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D016375:Antisense Elements (Genetics); D015153:Blotting, Western; D001921:Brain / Q000033:anatomy & histology; D001923:Brain Chemistry; D000939:Epitopes / Q000276:immunology; D005057:Eukaryotic Cells / Q000276:immunology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D015347:RNA Probes; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000276:immunology / Q000378:metabolism*; D014162:Transfection / Q000276:immunology
D000906:Antibodies; D016375:Antisense Elements (Genetics); D000939:Epitopes; D015347:RNA Probes; D011985:Receptors, Serotonin