Stereochemical approach to increase the specificity of steroid antibodies

Abstract

Most of the attempts to produce steroid antibodies have utilized one of the functional groups of the steroid molecule to form the corresponding haptene. We have prepared 19-hemisuccinate derivatives of androstenedione, 5α-dihydrotestosterone and testosterone, bound to BSA via the carbodiimide reaction to obtain antibodies with high specificity. With the hemisuccinate linked at the C-19 position the disturbance of the functional groups is reduced, introducing minimal changes and yielding the highest possible resemblance between the antigenic complex and the original steroid molecule. Furthermore, with the hemisuccinate chain in the axial position, the carboxyl group is located far enough away to avoid intramolecular interactions between the protein and the free functional groups. In this form the specificity improved significantly. After 12 weeks of injecting the haptenes into rabbits, we obtained the following antisera: for androstenedione at a dilution of 1:12000, with crossreactivity to androstanedione and epiandrosterone with a displacement effect around 50% of the 3H-androstenedione to dehydroepiandrosterone (DHEA) and testosterone was less than 25%; for dihydrotestosterone the antiserum titres were 1:6000 with crossreaction to testosterone, 5α-androstane 3–17-dione and androstenediol but less than 50% bound and minimal to DHEA and androstenedione; the testosterone antiserum in dilution 1:2000 revealed similar binding for dihydrotestosterone and very low for DHEA and androstenedione. We concluded: 1.—The ring D as well as the A of the steroid molecule were determinants in conferring specificity to the antibodies 2.—The coupling of the protein in the C-19 position near to the ring A together with 3-ketone group and the double bond in C-4 impeded the selective antibody production, decreasing specificity. In view of this 3.—The coupling length chain will be modified to achieve better specificity.