Development and optimization of an indirect enzyme-linked immunosorbent assay for 19-nortestosterone

Abstract

A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed, optimized and validated to measure this anabolic steroid. Infl uence of several physicochemical parameters, such as incubation time, ionic strength, detergent concentration and pH were selected to provide a highest sensitivity on the ELISA format. The regression equation of the fi nal inhibition curve was: y = -0.3194x + 1.6316, R 2 = 0.9927. The linear range was between 0.1 and 25 ng/ml and the IC 50 was 3.5 ng/ml. The specifi city was evaluated by fi ve structurally related anabolic steroids, and none of them had signifi cant cross-reactivity. Finally, the accuracy and precision of this assay were evaluated by means of spiked samples. The recovery was between 76.9 and 104.7%, and the variation coeffi cient was between 5.2 and 13.4%.