Considerations in the Measurement of Testosterone in Saliva and Serum Using ELISA Procedures

Background: ELISA procedures are widely available and used for the measurement of saliva and blood (serum and plasma) testosterone. Suggestions for strong correlations between these two fluids have been made but differences in assay format, as well as in the collection procedures, storage, and processing of samples can influence results.
Methods: The present study compared saliva and serum free testosterone concentrations in 20 healthy men (31.0±11.0 years; mean ± SD) using ELISA procedures. Men provided both a saliva and blood sample on the same day in the morning hours following an overnight fast. Special care was taken in the collection, storage, and processing of samples. Following complete thawing and mixing of samples, both fluids were analyzed in duplicate using commercially available ELISA kits, both prior to and following centrifugation.
Results: Saliva testosterone values were 440.6±238.2 pg∙mL-1 and 348.8±210.0 pg∙mL-1 without and with centrifugation, respectively. Serum testosterone values were 9.0±4.2 pg∙mL-1 and 8.3±3.7 pg∙mL-1 without and with centrifugation, respectively.
Conclusion: If utilizing ELISA procedures, saliva and serum testosterone values cannot be used interchangeably, at least when utilizing the ELISA procedures employed in the present study. This is evidenced by the approximate 10-100 times higher testosterone concentration in saliva as compared to serum. Moreover, processing samples via centrifugation leads to a significant (~23%) loss in testosterone in saliva, with a much smaller loss (~7%) in serum. Investigators and clinicians should take note of these findings if planning to measure saliva or serum testosterone using ELISA procedures.