Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody

Abstract

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11βHSD type 2. To assess 11β-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28pg/mL (77.7pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X−0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5–11.7% and 8.7–12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3nM at 08.00h and 5.1±3.6nM at 18.00h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22nM that increased to 8.6±1.2nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9nmol/day. In human volunteers following ingestion of green coffee bean extract for 2weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.