Determination of dehydroepiandrosterone sulfate in plasma by a one-step enzyme immunoassay with a microtitre plate

We have developed a rapid and cost-effective enzyme immunoassay for dehydroepiandrosterone sulfate (DHEA-S) in plasma, performed with samples on a microtitre plate within 2.5 h. No extraction or centrifugation steps are involved. The 3-hemisuccinate of dehydroepiandrosterone is labeled with horseradish peroxidase, then mixed with hydrogen peroxide substrate in the presence of the chromogen, tetramethylbenzidine. The detection limit of the assay is 12.5 pg of DHEA-S per well. Intra- and interassay CVs at three steroid concentrations (12.8, 1.28, and 0.16 mumol/L) ranged from 2.3 to 5.4% and 6.1 to 8.4%, respectively. Results correlated well (r = 0.95) with those of a radioimmunoassay with iodinated DHEA-S. The turnaround time for 41 samples (in duplicate) is 2.5 h, which includes 2 h of incubation time. The sensitivity of this one-step version and the linearity of its standard curve are equivalent to those of a less practicable two-step version. This technique may replace coated-tube enzyme immunoassays for routine use.