Development of a sensitive enzyme immunoassay for plasma and salivary steroids

Abstract

The development of an enzyme-labelled immunoassay (EIA) of sufficient range and sensitivity for determination of plasma and salivary steroids is described. The method is based on competition in the solid phase. A fixed amount of a specific antibody is immobilized on polystyrene beads, its amount being enough to bind about 50% of a steroid-enzyme conjugate (covalently linked steroid Jhorseradish peroxidase conjugate). The initial incubation at 25 degrees of serum sample or extract with the conjugate in presence of the immobilized antibody is followed by washing and the addition of a substrate for measurement of the enzyme activity either colorimetrically by the H(2)O(2)/o-phenylenediamine method or by the luminescence reaction based on the use of H(2)O(2)/luminol. The method is precise and accurate (CV < 10% for both the intra- and inter-assay studies). The sensitivity of the colorimetric determination is similar to that of radioimmunoassay, but the luminescence method is more sensitive. Radioimmunoassay and EIA gave results in good agreement (r > 0.97). The method has also been applied to salivary steroids: the procedure is direct and only a few mu1 of saliva are required. The levels of these steroids in saliva are only about a tenth of those in serum but directly correlated with them (r > 0.85). The enzyme immunoassay proposed is sensitive and precise enough to measure these steroids accurately in saliva and plasma.