Development of an Enzyme-Linked Immunosorbent Assay for Serum 11-Dehydrocorticosterone in Rat and Mouse

Abstract

We have developed an enzyme-linked immunosorbent assay (ELISA) for serum 11-dehydrocorticosterone (4-pregnen-21-ol-3,11,20-trione). The antiserum against 11-dehydrocorticosterone 21-hemisuccinate-conjugated bovine serum albumin was raised in rabbits. As an enzyme-labeled antigen, 11-dehydrocorticosterone 21-hemisuccinate was conjugated to horseradish peroxidase. Two ELISA systems were established: one without the extraction of steroids from serum (direct method), and another that used an HPLC purification step (HPLC method). The cross-reactivity of all steroids tested against the antibody was low except cortisone (92%); however, since cortisone levels in rats and mice are negligible, cortisone does not interfere with this direct ELISA. The measurable range of serum 11-dehydrocortiocosterone in both the direct and HPLC methods was 0.3-250 ng/ml and 0.78-400 ng/ml, respectively. Both methods displayed satisfactory parallel dilution, recovery and reproducibility; moreover, the values obtained with each method significantly correlated with the alternate method. To evaluate the two ELISA systems, the serum concentrations of 11-dehydrocorticosterone in normal rats and mice were determined by these two systems. The levels in Wistar rats fluctuated from 3 to 14 weeks of age (7.8+/-2.6 ng/ml) but at 1 week (1.7+/-1.2 ng/ml) were significantly low compared to other ages. No sex differences were found in rats and mice. Further, using the proposed direct method, chronological changes of rat serum 11-dehydrocorticosterone levels after 11-dehydrocorticosterone administration have been investigated together with corticosterone levels. These results verify that the proposed ELISA for 11-dehydrocorticosterone is useful for measuring 11beta-HSD activities in combination with the determination of serum corticosterone in rats and mice.