Immunogens for the preparation of retinoic acid antibodies

Abstract

The generation of antibodies to retinoic acid necessitates the prior synthesis of appropriate immunogens. This paper describes the synthesis and characterization of various retinoic acid immunogens. Immunogens were synthesized by reacting the carboxylic group of retinoic acid with bovine serum albumin, chicken IgG and human IgG; these immunogens are expected to produce antibodies against the ring portion of the retinoid molecule. For the generation of antibodies with specificity towards the retinoid polyene side chain and its carboxyl group, immunogens were prepared with an exposed retinoid polyene side chain and carboxyl group. Retinoic acid was derivatized to methyl-4-oxoretinoate, and carboxymethoxylamine attached via 4-oxo position to the B-ionone ring to yield methyl-4-oxoretinoic acid oxime, miz 402 for [M + H] +; E 1 cm 1% 1965 at 365 nm in 45% dioxane; the structure of the oxime was verified by NMR. The hapten was conjugated with bovine serum albumin, chicken IgG, human IgG, or with aminohexyl Sepharose 4B to produce various antigens. The p-hydrazinobenzoic acid derivative of 4-oxoretinoic acid was also used as a hapten. Immunogens were also prepared by conjugating retinoic acid to liposome-protein complexes. The prepared immunogens generated antiretinoic acid antibodies in mice and rabbits. The best immunogenic response was obtained with 4-oxoretinoic acid oxime-chicken IgG conjugate. Our work provides a systematic approach to synthesis of retinoic acid antigens and describes reproducible techniques for the preparation of retinoic acid haptens and retinoic acid-specific immunogens. The methods can be applied to the generation of immunogens to other retinoids and vitamins.