Assessment of urinary total testosterone production by a highly sensitive time‐resolved fluorescence immunoassay

Abstract

Radioimmunoassay (RIA) that involves purification of the analyte by organic solvent extraction is widely used. Although the extraction RIA is reliable when properly validated, it is time consuming and radioactive, we measure urinary total testosterone with a highly sensitive rapid and accurate time-resolved fluorescence immunoassay (TRFIA) method. High affinity antitestosterone antibody and Eu(3+) labeled Donkey antisheep IgG as tracers were used. The assay was evaluated for specificity, sensitivity, analytical recovery, precision and dilution linearity by the TRFIA method on urine samples. A satisfactory standard curve for testosterone TRFIA has been developed with good sensitivity (5.1 pmol/L). The validity of the assay for urinarytotal testosterone was confirmed by the good correlation between the results obtained by TRFIA (X) and those RIA (Y) (Y=0.075+0.971X, R=0.992). Specificity, analytical recovery, precision and dilution linearity studies were determined and all found to be satisfactory. Male urinary total testosterone excretion ranged from 64.00 to 374.11 nmol/24 hr, which was about four times more than the range for women urinary testosterone excretion (14.16-100.65 nmol/24 hr), which suggests that a direct, reliable, easy to automate, highly sensitive and specific TRFIA type assay for the measurement of total testosterone in urine samples has been developed.