Development of an enzyme immunoassay for serum 16-dehydropregnenolone

We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenol one (3 beta -hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuceinate conjugated bovine serum albumin (16-DHP-3HS-BSA) wa s raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was c onjugated to alkaline phosphatase. The minimal amount of 16-DHP detected wa s 4 pg (0.013 pmol)/assay and the measurable range was from 0.06-60 ng/ml ( 0.191-191 nmol/l). The intra-assay coefficient of variation (C.V.) was 4.1% (0.73+/-0.03 ng/ml, mean+/-S.D., n=6), and inter-assay C.V. was 7.7% (0.13 +/-0.01 ng/ml, n=6). A liner relation was observed between the serum sample dilution and the 16-DHP concentration. For the recovery study, authentic 1 6-DHP was added to a serum sample (original concentration: 0.10-0.14 ng/ml) , and the recovery was found to be 94.4-96.8% (final 16-DHP concentrations calculated: 0.29-16.3 ng/ml). To investigate the reliability of the present EIA, the values from our EIA were compared with those obtained by GC-MS. T he 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzy matically hydrolysed and then the 16-DHP measured by both methods. There wa s a good correlation between the levels determined by these methods (Pearso n's correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The ser um concentrations of 16-DHP in neonates and umbilical vein were 0.53+/-0.09 ng/ml and 0.88+/-0.61 ng/ml, respectively. No 16-DHP was detected in serum from normal healthy adults using the present EIA. These results suggest th at 16-DHP originates from the fetus and neonate.