Elsevier

Clinica Chimica Acta

Volume 364, Issues 1–2, February 2006, Pages 172-179
Clinica Chimica Acta

Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine

https://doi.org/10.1016/j.cccn.2005.06.019Get rights and content

Abstract

Background

Several methods are now available to estimate urinary testosterone levels that can only be performed in established big laboratories using GC/MS techniques. In clinical practice or for research projects, an inexpensive method that does not require skilled technicians would be useful. A simple, rapid and accurate ELISA method has been developed and applied in our laboratory to measure urinary non-conjugated and total testosterone.

Methods

High affinity anti-testosterone antibody and HRP-Donkey anti-sheep IgG (Horse Radish Peroxidase) as enzyme tracer were used to develop the ELISA method. The assay was evaluated for specificity, sensitivity, parallelism, accuracy and imprecision by the established methods on samples obtained from healthy volunteers. The results from the direct ELISA were compared to those after enzyme hydrolysis plus solvent extraction and HPLC or commercial kits.

Results

A satisfactory standard curve for the direct testosterone ELISA has been developed with good sensitivity. Cross-reactivity values of anti-testosterone antibody with major interfering steroids were minimal except for testosterone-3-glucuronide (58.8%). The validity of urinary testosterone assay was confirmed by the good correlation between the results obtained by the direct ELISA and those after enzyme hydrolysis and solvent extraction (Y = 0.987X + 0.398, R2 = 0.97). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Urinary testosterone excretion values obtained by our direct ELISA from healthy volunteers were generally in agreement with those published by other workers. Male urinary total testosterone excretion (non-conjugated and testosterone glucuronide) ranged from 177.9 to 865.3 nmol/day, which was about 3–6 times more than the range for women urinary testosterone excretion (34.5–308.8 nmol/day).

Conclusion

A direct, reliable, easy to perform, sensitive and highly specific ELISA type assay for the measurement of total testosterone in urine samples (conjugated and non-conjugated) has been developed. The novel features of the assay are that it does not require an initial extraction step or involve time consuming procedures such as chromatography. A simple method has also been developed to measure non-conjugated urinary testosterone excretion after solvent extraction alone.

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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