There is only one description of a homovanillic acid immunogen that was used successfully to generate polyclonal antibodies for use in immunoassays for this metabolite of dopamine [Gallacher and Ho (1992) Biogenic Amines, 9, 99-114]. Synthesis of an immunogen for homovanillic acid requires covalent conjugation of the acid to carrier protein and this necessarily involves alteration of the molecular structure of the acid. We designed a new homovanillic acid immunogen in which the haptenic determinants more closely resemble free homovanillic acid than did the conjugated homovanillic acid in the previously described immunogen. In the new immunogen (as in the earlier immunogen) homovanillic acid is linked to carrier protein through the 4-position of the metabolic. In the new immunogen, however, the 4-hydroxyl is replaced by an amide NH group, that resembles the OH group it replaces. The NH group is isosteric with an OH group and, like an OH group, can take part in hydrogen bonding interaction. The new immunogen was synthesised from 3-methoxyphenylacetonitrile. This was nitrated, and the 4-nitro product was isolated. The nitrile was then hydrolysed to the carboxylic acid which was protected as the methyl ester. The nitro group was then reduced to the amine and acylated with succinic anhydride thereby introducing the amide at the 4-position. The remaining carboxyl group from the succinimide linking moiety was coupled to carrier protein and the methyl ester protection was removed by hydrolysis to give the new homovanillic acid immunogen. When the new immunogen was used to generate antisera in sheep the resulting antisera exhibited higher titres than the earlier antisera and, more importantly, were more specific for homovanillic acid.
