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).