Antigenic complexes of steroid hormones formed by coupling to protein through position 7: Preparation from Δ 4 -3-oxosteroids and characterization of antibodies to testosterone and androstenedione

Abstract

A general method for rendering Δ 3-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7α-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17α-hydroxyprogesterone through the respective 4, 6-dienes. Immunization of rabbits with testosterone-7α-carboxymethyl-thioether-BSA and the homologous testosterone-7α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×10 9 1/mol) that showed little cross-reaction with androstenedione (< 1%) and with a variety of 17-oxoandrostane compounds (⩽ 0.5%). Conversely, immunization with androstenedione-7α -carboxyethyl-thioether-BSA yielded an antiserum with high affinity for androstenedione (Ka = 1. 04 × 10 10 I/mol) but minimal cross-reaction with testosterone (< 0.5%) and 17β-hydroxy-androstane compounds (⩽ 1%). The reaction of anti-testosterone and anti-androstenedione sera with their homologous haptens was not significantly inhibited by the closely related steroids 17α- epit estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5α-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5α-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%). A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.