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

Abstract

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (50O fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an llα-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an llα-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-¦O-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 > 0.98, n = 28) and saliva (r > 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 salivary testosterone concentrations in samples collected daily throughout one complete cycle ranged from 5O 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.