A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: Validation in human and mouse plasma |
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| Authors: | Min Sun Jenny Manolopoulou Ariadni Spyroglou Felix Beuschlein Constanze Hantel Zida Wu Maximilian Bielohuby Andreas Hoeflich Chao Liu Martin Bidlingmaier |
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| Affiliation: | 1. Department of Endocrinology, First Affiliated Hospital with Nanjing Medical University, No. 300 Guangzhou Road, 210029 Nanjing, China;2. Endocrine Research Laboratories, Medizinische Klinik – Innenstadt, Ludwig Maximilians University, Ziemssenstr. 1, 80336 Munich, Germany;3. Division of Clinical Endocrinology, Department of Medicine, Campus Charité Mitte, Universitaetsmedizin Berlin, Berlin, Germany;4. Laboratory of Mouse Genetics, Research Unit Genetics and Biometry, Research Institute for the Biology of Farm Animals – FBN, 18196 Dummerstorf, Germany;1. Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, China;2. Cadet Brigade, Third Military Medical University, Chongqing 400038, China;1. Department of Medicine, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand;2. Faculty of Agriculture and Life Sciences, Lincoln University, Canterbury, New Zealand;1. Department of Critical Care Medicine, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, AZ 85054, USA;2. Division of Cardiovascular Diseases, Mayo Clinic, Phoenix, AZ, USA;1. School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China;2. Shanghai Ninth People''s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200011, PR China;3. Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, United States;1. Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany;2. Department of Surgery, Klinikum der Universität München, Munich, Germany;3. Pediatrics Department, FMRP-USP, Ribeirão Preto, Brazil;4. Institut Cochin, Inserm U1016, Cnrs UMR8104, Université Paris Descartes, Paris, France;5. Clinical Biochemistry, Medizinische Klinik und Poliklinik IV, Munich, Germany |
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| Abstract: | BackgroundThe small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume.MethodsFollowing solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin–R–phycoerythrin (SA–PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown.ResultsThe assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1–15.6%/9.9–15.8% and testosterone 9.7–10.9%/7.7–11.4%) and correlate significantly to established assays (r = 0.94–0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6× NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261–396) pg/ml to 2008 (875–2467) in male and from 260 (210–576) to 1120 (734–1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4–8.3) ng/ml to 31.8 (30.4–33.9) ng/ml, P < 0.01).ConclusionsWe describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents. |
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