GENERATION OF ANTIPEPTIDE ANTIBODIES AGAINST SEROTONIN 5-HT2A AND 5-HT2C RECEPTORS

Anti-peptide antibodies were generated against several 13-17 amino aci d regions of rat serotonin 5-HT2A and 5-HT2C receptors. Peptides conta ining terminal cysteine residues were conjugated to bovine serum album in (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 carri er molecule were changed during the immunization schedule. For the ear ly immunizations, immunogens were BSA-peptides at ratios of 8-27 mol p eptide 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 p urified from sera (rabbits) or egg yolks (hens) using peptide matrices . Cell lines expressing similar densities of rat 5-HT2A or 5-HT2C rece ptors were used to monitor the specificity of purified antibodies on i mmunoblots 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 (t hree anti-5-HT2A and one anti-5-HT2C). None of the anti-peptide chicke n antibodies were useful on immunoblots or in immunocytochemistry. Sin ce there is a paucity of high affinity reagents selective for 5-HT2A o r 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. (C) 1997 E lsevier Science B.V.