Development and validation of a highly sensitive and specific enzyme immunosorbant assay for aldosterone: application to urine samples from cyp11b1 knockout mice

Abstract

The majority of immunoassays used to measure aldosterone (most potent mineralocorticoid) levels are still based on radio-iodinated tracer, lack sensitivity and specificity and there is a definite need for their improvement. The aim of this project is to develop a highly sensitive and specific ELISA method for urinary aldosterone estimation in samples obtained from wild type and cyp11b1 knockout mice. Antibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique (Innova Biosciences, Cambridge) and used to develop the ELISA method. Urine samples obtained from wild type and null mice were first hydrolysed with Helix Pomatia (Sigma), extracted with dichloromethane and reconstituted in assay buffer. Aliquots were then assayed using the ELISA technique previously published following some modifications to sensitise the assay (Al-Dujaili 2006, Clinica Chimica Acta 364 172-179). The aldosterone ELISA was validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivity with major interfering steroids was minimal: corticosterone=0.018%, cortisol=0.0014%, DOC=0.013% except for 5-dihydro-aldosterone=1.65%. Minimum detection limit of this ELISA was 2.2 pg/ml (6.2 pmol/l). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC separation step (Y=1.048X+0.006, R2=0.998, n=42). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels in male wild type and null mice on normal sodium diet were 42.710.3 (S.E.M.) pmol/g per day and 16.12.7 pmol/g per day, and on low sodium diet were 132.524.2 and 37.610.3 pmol/g per day, respectively.

In conclusion, a simple and highly sensitive ELISA has been developed to estimate urinary excretion of aldosterone and the assay can clearly confirm the animal's sodium intake status and distinguish between urinary aldosterone levels in wild type and null mice.