A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva.

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (500 fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an 11 alpha-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an 11 alpha-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the "enzyme label", and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by "extracting" testosterone from samples with a solid-phase anti testosterone-3-/0-carboxymethyl/-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r greater than 0.98, n=28) and saliva (r greater than 0.99, n=28). In saliva samples collected at 2 hourly intervals by normal healthy women (n=5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n=7), mean daily throughout one complete cycle ranged from 50 to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50%, and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.