Enzyme immunoassay of serum cortisol using a new transferable needle lid technique

A simple enzyme immunoassay of serum cortisol using for the first time a transferable needle lid as solid phase has been developed. The needles coated with second antibody and dipped into the wells of a microtitre plate bind the specific antibody of a competitive enzyme immunoassay mixture. Bound enzyme activity is estimated in the wells of another microtitre plate. This technique provides further advantages on the frequently used microtitre plate version. Washes between the immunological and the enzymatic reaction take very short time and are less laborious. Due to the facility of simultaneous starting and stopping of all reactions, a better precision and sensitivity is achieved. In the present cortisol assay, horseradish peroxidase covalently coupled to cortisol-21-hemisuccinate was used as enzyme label and tetramethylbenzidine as the chromogen for measuring enzyme activity. No extraction or deproteinization steps are involved. The turn around time for 41 samples (in duplicate) is 2.5 h. The detection limit of the assay is 5 pg of cortisol per well. Results of the present method correlated well (r = 0.92) with those of a commercial radioimmunoassay using iodinated cortisol.