DIRECT ENZYME-IMMUNOASSAY FOR PLASMA ANDROSTENEDIONE

A competitive and sensitive enzyme-linked immunoadsorbent assay (ELISA) allowing determination of androstenedione (A) in unextracted human plasma is described. A highly specific antiserum (anti-4-androsten-11-alpha-ol-3,17 dione-11-alpha hemisuccinate-bovine serum albumin) was employed. Horseradish peroxidase (HRP) was used as the label enzyme; free-bound separation was carried out by adsorbing purified IgG of antiserum on polystyrene balls. Plasma protein binding capacity was reduced by adding 8-anilinonaphtalene-1-sulfonic acid ammonium salt (ANSA). After 3h-incubation, enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride as chromogen and hydrogen peroxide as substrate. The sensitivity of the assay was 5 pg/tube. In order to compare ELISA with radioimmunoassay (RIA) A estimations, plasma samples from thirty women with different endocrine disorders were assayed. A good correlation was found between the two techniques: r = 0.930, p. < 0.001. The accuracy, reproducibility and sensitivity of this procedure make it ideal for rapid plasma A determination for clinical studies.