The radioimmunoassay of pregnanetriol 3 alpha-glucuronide

The hapten (5 beta-pregnane-17,20 alpha-diol-3 alpha-yl glucuronide) required to develop the radioimmunoassay was synthesized by an unambiguous chemical synthesis. An immunogenic complex was synthesized by coupling bovine serum albumin (BSA) to the glucuronide by mixed acid anhydride reaction. The immunogenic complex was used to induce the formation of specific antibodies in rabbits. In addition, the required radioligand [6,7-3H]5 beta-pregnanetriol 3 alpha-glucuronide was prepared with appropriate specific radioactivity. The characteristics of the antibody were determined with regard to specificity and sensitivity and the precision and accuracy of the assay method established. As applied to urine samples this method is superior to existing procedures in that they avoid the hydrolysis step and can be used directly on diluted urine. Examples have been given of useful application of this technique in clinical practice.