Testosterone analysis with a saliva ELISA

Enzyme immunoassay is performed for the quantitative estimation of free testosterone in human saliva.

Testosterone measurement is used in the diagnosis and treatment of disorders concerning the male sex hormones (androgens), such as primary and secondary hypogonadism, as well as male impotence, and in female hirsutism (excessive hair), virilization (masculinization) because of tumors, polycystic ovaries and adrenogenital syndrome.

  • Method — ELISA
  • Regulatory status — EU: CE
  • Specimen/volumes — 50 µL saliva/without extraction
  • Kit size — 12 x 8
  • Substrate/isotope — TMB 450 nm
  • Incubation time — 1 x 2 hours, 1 x 30 minutes
  • Standard range — 10–900 pg/mL

The enhanced IBL International Testosterone Saliva ELISA is calibrated to the LC-MS/MS reference technique and comprises interchangeable reagents within the IBL saliva ELISA product series.

The Testosterone Saliva ELISA can be automated without difficulty on open platforms such as the EVOlyzer® from Tecan.

Benefits of the Testosterone Saliva ELISA

  • High sensitivity
  • Calibration to the LC-MS/MS
  • Streamlined pipetting protocol
  • Alignment to entire Saliva line
  • Optimization for automation

ELISA vs mass spectronomy

Mass spectrometry (MS) is said to be the reference approach for steroid hormone quantification. This method does however signify a large investment and therefore is not found in all laboratories.

All the IBL saliva assays have been made to correlate to this reference approach, and even more notably, they only necessitate low sample volume for assessment of the steroid hormones.

Consistent and reliable processing within Saliva product line

Tecan’s Saliva ELISA product line (progesterone, cortisol, testosterone and DHEA) can be processed in a comparable way and effortlessly carried out in combination:

  • The TMB, stop solution, and wash solution consists of the same design within DHEA, cortisol and progesterone saliva ELISA (RE52651, RE52611 and RE52281)*
  • Only 50 µL sample volume is needed
  • The enzyme conjugate, and the substrate and stop solutions are evenly pipetted with 100 µL
  • The kits have as few as six standards and two controls
  • All the kits employ the same sample diluent buffer.

*Valid lot numbers of TMB, stop solution and wash solution are mentioned on the outer labels and Quality Control certificates of the kits.


The IBL International Testosterone Saliva ELISA (RE52631) is versatile and can be used on various ELISA processors. Open automated systems such as the EVOlyzer® from Tecan have been tested and exhibit good correlation to the manual operation.

Testosterone analysis with a saliva ELISA

Image Credit: Tecan

The joint use of assays, process script and automate has to be confirmed separately on site by each lab.

Applications of free testosterone determination

Testosterone is a steroid hormone, which forms in the testes of males and in smaller amounts in the ovaries of women. In both women and men, a considerable amount is created by the conversion of circulating DHEA, DHEA-S and androstenedione. It exhibits a diurnal rhythm, with maximum levels in the morning and the lowest amount by about midnight.

Testosterone has a vital role to play in the development of male reproductive tissues; supports bone mass, increased muscle and hair growth; and is crucial for health, cardiovascular health, sexual function, stamina and a healthy immune system.

Testosterone measurement is used in the diagnosis and treatment of disorders concerning androgens, the male sex hormones. These comprise primary and secondary hypogonadism, disorders stemming from testicular difficulties or deficiencies of the pituitary glands or hypothalamus.

Additionally, it also helps investigate impotence in males and females, hirsutism (excessive hair), polycystic ovaries virilization (masculinization) because of tumors and adrenogenital syndrome. Testosterone along with cortisol levels also signifies a valuable parameter in sports medicine and stress research.

Measurement of testosterone in saliva

About 1%–5% of testosterone is unbound from plasma proteins and thus biologically active. It is discharged through the salivary glands and mostly converted to 5a-dihydrotestosterone. The testosterone concentration in saliva reflects the quantity of free testosterone in plasma.

Sampling time

There is no exception in the case of testosterone within steroid hormones as concentrations have a diurnal rhythm. Therefore, the number of samples and the sampling time are decisive in a clinical setting to consistently assess the peak and baseline level of testosterone independently.