STUDIES ON A RADIOIMMUNOASSAY FOR PLASMA TESTOSTERONE

Abstract

Using one step of Sephadex LH-20 micro column chromatography with the soluvent system of hexane, benzene, methanol (90:5:5), complete separation of testosterone from 5α-dihydrotestosterone (DHT) was developed. The antiserum against testosterone-3 oxime-bovine serum albumin was supplied by the Department of Pharmacological Research, Teikoku Hormone Mfg. Co., Ltd. and was used at a dilution of 1:20, 000. The antiserum has high affinity for testosterone and DHT (Cross reaction: testosterone 100, DHT 117%). It is possible to determine simulteneously testosterone and DHT concentrations in plasma by this method.
Plasma of 0.05ml from men and 0.5ml from women was used for analysis. Following extraction with ether and chromatography on Sephadex LH-20, the dried purified extract was incubated with antiserum at room temperature for 30min. (NH₄)₂SO₄ was used to separate free from bound testosterone.
Various factors affecting the method blank, accuracy, sensitivity, and precision were investigated. The mean blank was 9.0±5.9 (M±SD) pg per sample. The mean recovery of testosterone ³H added to 21 samples was 83.6±6.3%. The accuracy and precision of the method were satisfactory. The concentration of testosterone in plasma from men was 559±170ng/dl, and in the sample collected from women 32±12ng/dl, and in plasma from patients with carcinoma of the prostate who were treated by synthetic estrogenic agents for 1 to 7 years 19±6ng/dl. Our values for plasma testosterone concentration in women were lower than those reported previously. To determine plasma testosterone concentration by the use of radioimmunoassay, complete separation of testosterone from other steroids is quite essential.