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

Clin Chim Acta. 2006 Feb;364(1-2):172-9. doi: 10.1016/j.cccn.2005.06.019. Epub 2005 Aug 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antibody Specificity / immunology
  • Chromatography, High Pressure Liquid
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Female
  • Glucuronides / chemistry*
  • Humans
  • Immune Sera / immunology
  • Male
  • Middle Aged
  • Reference Standards
  • Reproducibility of Results
  • Testosterone / chemistry
  • Testosterone / standards
  • Testosterone / urine*


  • Glucuronides
  • Immune Sera
  • Testosterone