ELISA procedure

The commercially available, competitive ELISA is based on the competition between cortisol and cortisol–acetylcholinesterase (AChE) conjugate for the anticortisol antibody binding sites. Particularly, the assay was performed by the addition of cortisol standards/samples, AChE, and the cortisol-specific, mouse monoclonal antibody in the wells. During the incubation (overnight at + 4 °C), the antibody–cortisol (either free or tracer) complex bound to the goat polyclonal anti-mouse IgG captured on the solid phase. After a washing step, the bound enzyme conjugate was revealed thanks to the addition of the Ellman’s reagent (which contains the substrate) producing a yellow product. A microplate reader Benchmark BIO-RAD (Hercules, CA, USA) was used to measure the absorbance at 405 nm. This latter was inversely proportional to the amount of free cortisol present in the well during the incubation. The results were expressed in percentages of the maximum absorbance (B/B₀%) using Eq. 1:

The cross-reactivities of steroids in the selected ELISA kit are as follows: cortisol 100%, dexamethasone 15%, prednisolone 4.0%, cortexolone 1.6%, 11-deoxycorticosterone, and 17-hydroxyprogesterone 0.23%, cortisol glucuronide 0.15%, corticosterone 0.14%, cortisone 0.13%, and finally androstenedione, enterolactone, estrone, 17-hydroxypregnenolone, pregnenolone, and testosterone < 0.01% (data from manufacturer).