Generation of anti-peptide antibodies against serotonin 5-HT2A and 5-HT2C receptors.
Anti-peptide antibodies were generated against several 13-17 amino acid regions of rat serotonin 5-HT2A and 5-HT2C receptors. Peptides containing terminal cysteine residues were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) with the cross-linking reagent sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate). Both the carrier protein and the number of peptide molecules per carrier molecule were changed during the immunization schedule. For the early immunizations, immunogens were BSA-peptides at ratios of 8-27 mol peptide per mol BSA. For the later boosts, immunogens were OVA-peptides at ratios of 1-2 mol peptide per mol OVA. The peptide constructs were used to immunize rabbits and chickens. Anti-peptide antibodies were purified from sera (rabbits) or egg yolks (hens) using peptide matrices. Cell lines expressing similar densities of rat 5-HT2A or 5-HT2C receptors were used to monitor the specificity of purified antibodies on immunoblots and in immunocytochemistry. A total of five out of the six rabbit antibodies were positive on immunoblots (three anti-5-HT2A and two anti-5-HT2C) and four were also positive in immunocytochemistry (three anti-5-HT2A and one anti-5-HT2C). None of the anti-peptide chicken antibodies were useful on immunoblots or in immunocytochemistry. Since there is a paucity of high affinity reagents selective for 5-HT2A or 5-HT2C receptors, these rabbit antibodies will be useful tools. The methods used to generate site-directed antibodies specific for 5-HT2A or 5-HT2C receptors should be applicable to other proteins.
Backstrom
J R
JR
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6600, USA. BackstJR@ctrvax.Vanderbilt.edu
Sanders-Bush
E
E
D000818:Animals; D000917:Antibody Formation; D001923:Brain Chemistry / Q000502:physiology*; D002645:Chickens; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D009479:Neuropeptides / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000276:immunology*
D009479:Neuropeptides; D011985:Receptors, Serotonin