Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine
Introduction
Testosterone is one of the most important androgens secreted into the blood and is produced primarily by the testes in men and the ovary/peripheral conversion from DHEA in women [1], [23]. Several researchers have emphasised the importance of testosterone analysis in urine for androgen related disorders in men and women [2], [3], [4], [5], [6], [7], [23]. The majority of methods available to estimate urinary testosterone excretion are based on GC/MS techniques that involve multi-step procedure such as hydrolysis, extraction, derivative formation and purification [2]. While GC/MS methods are accurate, suitable for the estimation of many steroid hormones simultaneously and can serve as reference to other assays, they do require time, skilled personnel and expensive equipment [8], [9], [10], [11], [12], [13], [14]. HPLC techniques have also been used to estimate urinary testosterone [9], [15]. Various immunoassay methods have been developed to measure testosterone levels in plasma [14], [16], [17], [18], [19], however, the measurement of this steroid hormone alone in urine samples did not actually receive much attention, except for some radio-immunoassays designed for serum or plasma testosterone and have been modified for urine samples [20], [21], [22]. The measurement of testosterone in urine (a clinically important steroid hormone) offers useful information as it can give a good indication of the amount of the hormone produced over 24 h [2], [23]. The assay has to be quick, simple, routine and can determine total testosterone produced (conjugated and non-conjugated). ELISA methods are now widely used for routine estimation of steroid hormones [24], [25], [26].
We describe here the development and validation of a simple and direct ELISA method to measure urinary testosterone excretion without hydrolysis and solvent extraction. Non-conjugated urinary testosterone excretion can also be estimated after a simple solvent extraction step.
Section snippets
Assay reagents
Anti-testosterone antibodies raised in sheep were purchased from Micropharm, London, UK, and used at a dilution of 1:200,000.
Enzyme tracer (HRP-Donkey anti-sheep IgG antibody) was purchased from Micropharm, London and used at a dilution of 1:1000.
Testosterone standard (purum, 86500) was purchased from Sigma-Aldrich, Poole, Dorset, England. All other steroid hormones used for cross-reactivity studies (see Table 1) were obtained from Sigma-Aldrich, Poole, Dorset, England and Straloids, Newport,
Assay protocol for the direct urinary testosterone ELISA
Testosterone was measured using an indirect ELISA technique. We employed a modification of the methods described elsewhere [26], [27]. All samples were assayed in duplicate; 96-well ELISA plates were coated with testosterone-3-CMO-BSA conjugate in coating buffer at a concentration of 2 μg/ml by pipetting 200 μl into each well using a multi-Channel pipette, Biohit Plc, Finland. The plates were then covered with parafilm and left overnight at 4 °C. The contents of the plates were then discarded
Optimisation of testosterone standard curve
Standard curves performed according to the direct urine assay protocol and carried out in assay buffer or 0.2 M urea solution were almost identical, and therefore, buffer standard curves were used through out. Typical standard curve data for testosterone ELISA are shown in Fig. 1. Sensitivity (minimum detection limit) for the testosterone ELISA standard curve was determined according to the method of Abraham [33] in which it represents the amount of testosterone that produced a statistically
Discussion
A direct, reliable, easy to perform, sensitive and specific ELISA type assay for the measurement of conjugated (glucuronide) and non-conjugated testosterone in urine samples has been developed and validated. The assay enables the scientist to process a large number of samples within a short period of time and does not require highly skilled personnel [32]. The novel features of the assay are that it does not require an initial extraction step or involve time consuming procedures such as
Acknowledgements
This work was partly funded by the World Anti-Doping agency, and the author is grateful for the technical support of Mr. David Aitkinson.
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Dr. Emad Al-Dujaili was partly funded by the World Anti-Doping agency.