Development of an enzyme immunoassay for serum 16-dehydropregnenolone.
We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenolone (3beta-hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuccinate conjugated bovine serum albumin (16-DHP-3HS-BSA) was raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was conjugated to alkaline phosphatase. The minimal amount of 16-DHP detected was 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 16-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. The 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzymatically hydrolysed and then the 16-DHP measured by both methods. There was a good correlation between the levels determined by these methods (Pearson's correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The serum 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 that 16-DHP originates from the fetus and neonate.
Tagawa
N
N
Clinical Chemistry Laboratory, Kobe Pharmaceutical University, Japan.
Saiki
K
K
Kobayashi
Y
Y
D000328:Adult; D000469:Alkaline Phosphatase / Q000276:immunology; D000918:Antibody Specificity; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005312:Fetal Blood / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007124:Immunoenzyme Techniques; D007231:Infant, Newborn; D011247:Pregnancy; D011284:Pregnenolone / Q000031:analogs & derivatives* / Q000097:blood* / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000276:immunology
D012710:Serum Albumin, Bovine; C003592:16-dehydropregnenolone; D011284:Pregnenolone; D000469:Alkaline Phosphatase