ABSTRACT
A detailed method for measuring aldosterone in serum/plasma is described. Aldosterone is first extracted into dichloromethane to improve the specificity of the assay. A radioimmunoassay is performed on the dried extract using an antibody raised in rabbits to aldosterone conjugated at position C3, and iodinated aldosterone. Details are given for optimizing conditions for the second antibody, raised to rabbit immunoglobulins, and carrier protein used in the separation of the antibody bound fraction from the free aldosterone at the end of equilibration. Details are provided in additional notes, for steps in the assay that could be problematic.
Subject(s)
Aldosterone/blood , Radioimmunoassay/methods , Aldosterone/analysis , Aldosterone/isolation & purification , Animals , Humans , Sensitivity and SpecificityABSTRACT
Measurement of angiotensin 1, released in plasma by the action of endogenous enzyme renin on endogenous substrate, angiotensinogen is described. Details are given for the generation of angiotensin 1 from plasma using controlled conditions of pH and temperature. A radioimmunoassay to quantify the generated material is then described using anti-angiotensin 1 antibody and iodinated angiotensin 1 as label. Separation of the antibody-bound fraction from the free is achieved using dextran-coated charcoal. Problems of cryoactivation of prorenin and the labile nature of angiotensin 1 are highlighted. Additional notes describe steps in the assay that are critical.
Subject(s)
Radioimmunoassay/methods , Renin/analysis , Renin/blood , Angiotensin I/analysis , Angiotensin I/blood , Animals , Enzyme Activation , Humans , Substrate SpecificityABSTRACT
BACKGROUND: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. METHODS: We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. RESULTS: Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. CONCLUSIONS: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.
