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17beta-Hydroxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-3H] 17beta-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-3H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminum sheets and the steroid containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from immature male rainbow trout contain ca 0.2-0.4 mature, serum levels rise sharply to reach values of 2 to greater than 9 microng/100 ml of 17beta-hydroxyandrost-4-ene-3,11 dione. As male fish mature, serum levels rise sharply to reach values of 2 to greater than 9 microng/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels (ca 0.2 microng/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.