A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative

An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.