Meeting Abstract
90.1 Friday, Jan. 7 How to tell when your fecal assay is crap: considerations for development and validation of fecal steroid assays HARRIS, B.N.*; SALTZMAN, W.; DE JONG, T.R.; PEREA-RODRIGUEZ, J.P.; CHAUKE, M.; MILNES, M.R.; Univ. of Calif., Riverside, CA; Univ. of Calif., Riverside, CA; Univ. of Calif., Riverside, CA; Univ. of Calif., Riverside, CA; Univ. of Calif., Riverside, CA; Institute for Conservation Research, Escondido, CA bharr002@ucr.edu
Fecal hormone assays provide many advantages (e.g. repeated, non-invasive collection; time-integrated results); however, thorough validation is imperative to ensure that data are reliable, repeatable and biologically relevant. Steroid hormones circulate in the native or parent form, but, due to catabolism by both the liver and gut microorganisms, are excreted as a collection of metabolites. Thus, use of an appropriate antibody, as well as sampling method and time course, is essential for obtaining valid results. We have characterized fecal glucocorticoid (GC) concentrations in California mice (Peromyscus californicus) using three different antibodies: a GC metabolite antibody (5α-pregnan-3β,11β, 21-triol-20-one; Mostl & Palme, Vienna, Austria), and two polyclonal corticosterone (B) antibodies (CJM06, C. Munroe, Davis, CA; 07120113, MP Biomedicals, Solon, OH). Fecal GCs were successfully detected using all three antibodies; however, concentrations and assay consistency varied. Using a combination of laboratory methods (HPLC, RIA, EIA) and in-vivo manipulations (3H-B injection, diurnal rhythm assessment, ACTH injection, acute and chronic stress) we have shown that fecal GCs do not always mirror changes in plasma B. Conflicting results may indicate lack of antibody sensitivity for the species-typical metabolites, or that changes in plasma B levels were too small or transient to be reflected in fecal GCs. The antibodies tested have been used, but not necessarily systematically validated, in a wide range of species (including several rodents), reinforcing the importance of validating assays for each species; a mouse is not just any mouse hormonally speaking.
