A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: validation in human and mouse plasma.

The small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume. Following solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin-R-phycoerythrin (SA-PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown. The assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1-15.6%/9.9-15.8% and testosterone 9.7-10.9%/7.7-11.4%) and correlate significantly to established assays (r=0.94-0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6 × NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261-396) pg/ml to 2008 (875-2467) in male and from 260 (210-576) to 1120 (734-1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4-8.3) ng/ml to 31.8 (30.4-33.9) ng/ml, P<0.01). We describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.