Development and validation of highly sensitive and specific enzyme immunosorbant assays for deoxycorticosterone and corticosterone: application to urine samples from cyp11b1 knockout mice

Abstract

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone. Cyp11b1 encodes 11_-hydroxylase which catalyses the conversion of DOC to B in rodents. The aim of this study is to develop sensitive and specific ELISA methods to estimate urinary DOC and B levels in mice. Antibodies against DOC and B were raised in rabbits by our laboratories as previously described and HRP-Goat anti-Rabbit IgG enzyme tracer (Upstate, UK) were used to develop the ELISA methods. Urine samples obtained from wild type and null mice were first hydrolysed with Helix Pomatia, extracted with dichloromethane and reconstituted in assay buffer. Aliquots were then assayed using the ELISA technique previously published (Al-Dujaili 2006, Clinica Chimica Acta. 364: 172-179). The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivity with major interfering steroids was minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (11-dehydro-B=0.006%, cortisol=0.016%, DOC=0.04% and aldosterone=0.14%). Minimum detection limit for DOC ELISA was 2.8 pg/ml (8.5 pmol/l), and for B ELISA was 12.2 pg/ml (0.035 nmol/l). The validity of urinary DOC and B ELISAs were confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC separation step (DOC ELISA: Y=1.092X-0.012, R2=0.988, n=42; B ELISA: Y=1.047X-0.226, R2=0.996, n=42). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using these assays: mean urinary DOC and B levels in female wild type mice were 0.1960.033 (S.E.M.) and 65.3813.04 nmol/g per day and in null mice were 8.561.27 and 3.6610.48 nmol/g per day respectively. In male mice, DOC and B levels in wild type were 0.0480.14 and 6.060.87 nmol/g per day versus 1.280.298 and 0.6410.112 nmol/g per day in null animals. In conclusion, simple, rapid and sensitive ELISAs have been developed to estimate urinary excretion of DOC and B and the assays can clearly distinguish between urinary steroid excretion of wild type and null mice.