Development and Validation of a Fecal Testosterone Biomarker: RESULTS(1)


Assay Validation

The cross-reactivity of several steroids with the polyclonal antibody shows very high specificity for testosterone. The 5-reduction product of testosterone, 5-alpha-di-hydrotestosterone (5a-DHT), resulted in 57% cross-reactivity; the next highest cross-reactivity, 0.30%, was shown by androstenediol. Androsterone, dehydroepiandrosterone, estradiol, progesterone, and pregnenolone all had cross-reactivities of less than 0.04%. The bile acids had reactivities of less than 0.02%. The slopes generated from the serially diluted samples to determine parallelism were not different from the slope of the standard curve (p > 0.1). In the final cross-reactivity experiment in which a nonradioactive fecal sample was separated on HPLC and fractions were analyzed using the ELISA, the only immunoreactive peak occurred in fractions coinciding with the UV peak for testosterone.

The detection limit of the ELISA was less than 3 pg/ well. The average interassay coefficients of variation for the three concentrations of testosterone were 10.5%, 12.4%, and 11.0% at, respectively, 66%, 51%, and 21% displacement of labeled testosterone. The intraassay coefficients of variation for the fecal extract were 8.04%, 8.37%, and 4.52% at, respectively, 40%, 50%, and 75% displacement.

This entry was posted in Testosterone and tagged Biomarker, Mus musculus, Peromyscus maniculatus, Testosterone.