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Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

By Emad A S Al-Dujaili, LJ Mullins, MA Bailey and CJ Kenyon


Background: Clinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases. Methods: 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. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones. Results: Cross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R2 = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice. Conclusion: We describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia. © 2009 Elsevier Inc. All rights reserved

Year: 2009
OAI identifier: oai:eresearch.qmu.ac.uk:706

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  1. (1988). A competitive microtitre plate enzyme immunoassay for plasma aldosterone using a monoclonal antibody. doi
  2. (2008). A highly sensitive immunofluorometric assay for the measurement of aldosterone in small sample volumes: validation in mouse serum. doi
  3. (2004). A quick glance at rapid aldosterone action. Molecular and Cellular Endocrinology., doi
  4. (1974). A simple radioimmunoassay for urinary aldosterone.
  5. (2006). Aldosterone assays: an urgent need for improvement. Clin Chemistry., doi
  6. (1978). Aldosterone diagnosis in hypertension: comparative evaluation of radioimmunoassay for urinary aldosterone and 18-OH-corticosterone. Klin Wochenschr., doi
  7. (1985). Analysis of steroids. In gas capillary chromatography in clinical medicine doi
  8. (2006). Automated chemiluminescence-immunoassay for aldosterone during dynamic testing: comparison to radioimmunoassays with and without extraction steps. Clin Chem., doi
  9. (1985). Development and application of a direct radioimmunoassay for aldosterone in saliva. Steroids doi
  10. (1981). Development and application of a simple radioimmunoassay for urinary aldosterone. Clinica Chimica Acta., doi
  11. (2006). Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated Testosterone excretion in urine. Clinica Chimica Acta., doi
  12. (2008). Development and validation of highly sensitive and specific enzyme immunosorbant assays for deoxycorticosterone and corticosterone: application to urine samples from cyp11B1 knockout mice. Endocrine Abstracts,
  13. (1960). Double isotope derivative assay for aldosterone in biological extracts.
  14. (1990). Enhanced chemiluminescent immunoassay for aldosterone. doi
  15. (2000). Enzymatic hydrolysis of conjugated steroid metabolites: search for optimum conditions using response surface methodology. Analyst, doi
  16. (1987). Enzyme-linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone., Clin Chim Acta. doi
  17. (1990). Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies. doi
  18. Estimation of 24-h aldosterone secretion in the dog using the urine aldosterone:creatinine ratio. doi
  19. (1994). High incidence of primary aldosteronism in 199 patients referred with hypertension. Clin Exp Pharmacol Physiol., doi
  20. (2001). Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from helix pomatia and ampularia: determination of urinary cortisol and is metabolites. Steroids, doi
  21. (1985). Hydrolysis of steroid conjugates.
  22. (2006). its sex dependence in rats. doi
  23. (1995). Methods for urinary testosterone analysis. doi
  24. (1981). Optimisation of a direct radioimmunoassay for plasma aldosterone: doi
  25. (2006). Plasma aldosterone: Comparison between chemiluminescence-based and RIA methods Clin Chemistry., doi
  26. (1999). Potentially high prevalence of primary aldosteronism in a primary-care population. Lancet, doi
  27. (2000). Prevalence of primary aldosteronism among Asian hypertensive patients in Singapore. doi
  28. (1999). Primary aldosteronism: a common and curable form of hypertension. Cardiol Rev., doi
  29. (1992). Primary aldosteronism: hypertension with a genetic basis. Lancet, doi
  30. (1995). purification and measurement of steroids by HPLC, and GC-MS. doi
  31. (1973). Radioimmunoassay of aldosterone in plasma and urine: Validation of a novel separation technique. Clin Sci Mol Med.,
  32. (1974). Radioimmunoassay of steroids in biological materials. Acta Endocrinol.,
  33. Serozyme: a manual non-isotopic immunoassay system: in Immunometric assays; overview & new developments.
  34. (1969). Solid-phase radioimmunoassay for oestradiol-17β.
  35. (1978). The development and application of a direct immunoassay for plasma aldosterone using 125I-labelled ligand; comparison of three methods,
  36. (2005). The new biology of aldosterone.
  37. (1982). The renin-angiotensinaldosterone system.

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