Abstract
Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7α-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7α-CETE-KLH) or with 17-OHP-3-(
O-carboxymethyl)oxime-KLH (17-OHP-3-CMO -KLH). The resultant antisera were assessed using [
3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 × 10
91/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7α-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10–20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO -KLH were further assessed using [
125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 × 10
101/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.