Corticosterone and Testosterone Assays
In the first experiment, we quantified baseline and stress-induced plasma corticosterone concentrations using an Arbor Assays Corticosterone ELISA Kit (Cat#: ADI-900-065). Samples were run in duplicate at a 1:50 dilution following treatment with 1% steroid displacement buffer. Each plate contained a standard curve run in triplicate. Absorbance was measured at 405 nm using a SpectraMAX 190 spectrophotometer (Molecular Devices). We compared the mean value of the duplicates for each sample to a standard curve that contained known amounts of corticosterone. A 250 pg/mL standard was run in triplicate on each plate. The intra-assay coefficient of variation and inter-assay coefficient of variation were 12.8% and 14.0% respectively. Cross-reactivity of the testosterone antibody was as follows: androstenedione 7.2%, estradiol 1%, dehydroepiandrosterone 1%, dihydrotestosterone 1%, and progesterone 1% (Enzo Life Sciences).
In the second experiment, baseline plasma corticosterone concentrations were measured in duplicate following standard radioimmunoassay techniques (Wingfield et al., 1992). To determine the coefficient of intra- and inter-assay variation, four standard samples were prepared with 200 pg of corticosterone and plasma and standard sample tubes were prepared with 500 μl ddH2O and 2000 dpm of tritiated corticosterone (NET-399) from PerkinElmer Life Sciences, Inc. (Boston, MA, USA). Samples were equilibrated overnight at 4 °C. Corticosterone was extracted from plasma using 4 ml of diethyl ether and dried in a 37 °C water bath with the aid of nitrogen gas. Following extractions, samples were suspended in 500 μl of phosphate buffered saline and refrigerated overnight at 4 °C. The following day, we determined individual extraction efficiency using 50 μl of each sample. Corticosterone concentrations were corrected for individual extraction efficiency (mean recoveries were 83%). For the assay, each sample was allocated into two duplicates, each consisting of 200 μl. 100 μl of corticosterone antibody (B3-163; Endocrine Sciences, Calabasas, CA, USA) and 100 μl of tritiated corticosterone were added to each sample and standard tube. We compared the mean value of the duplicates for each sample to a standard curve (also run in duplicate) that contained known amounts of corticosterone (C2505 corticosterone standard, Sigma-Aldrich, St. Louis, MO, USA). The intra-assay coefficient of variation and inter-assay coefficient of variation were 11.4% and 13.3% respectively.
Testosterone was only measured in the second experiment, in which plasma samples were collected from focal male zebra finches 4 days prior to and 3 days following experimental treatment (LPS-focal: cue of disease, saline-focal: no cue of disease). Plasma testosterone concentrations were measured using an Enzo Testosterone ELISA Kit (Cat#: ADI-900-065). Samples were run in duplicate at a 1:20 dilution following treatment with 1% steroid displacement buffer. Each plate contained a standard curve run in triplicate. Absorbance was measured at 405 nm using a SpectraMAX 190 spectrophotometer (Molecular Devices). The intra-assay coefficient of variation and inter-assay coefficient of variation were 12.8% and 14.0% respectively. Cross-reactivity of the testosterone antibody was as follows: androstenedione 7.2%, estradiol 1%, dehydroepiandrosterone 1%, dihydrotestosterone 1%, and progesterone 1% (Enzo Life Sciences).