Hormone assay

CORT assays were conducted with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences 900-097) that has been widely used to determine plasma CORT concentrations across multiple taxa (Hopkins and Durant 2011; Klukowski 2011; Rivers et al. 2014). The kit was tested through parallelism between a serial dilution of a pooled sample of lizard plasma and a serial dilution of standard stock solution. We determined that an optimal dilution of plasma was 1:30, which is congruent with other studies using small sample volumes (Phillips and Klukowski 2008; Klukowski 2011).

Plasma aliquots of 10 µL were combined with 10 μL of steroid displacement reagent (SDR) and incubated for 10 min. Then, 280 μL of assay buffer were added to create a 30-fold dilution. Samples for each individual were run in duplicate and averaged. Six out of 70 blood samples contained less than 10 μL of plasma and were subsequently assayed without duplication. Instead, plasma volumes of 3.33 μL were run in singlet and kept at a 1:30 dilution with assay buffer and SDR. Excess reagent was washed away with a 5% p-nitrophenyl phosphate solution. The enzyme reaction was stopped after 60 min, and the light absorbance was read at 405 nm using a spectrophotometer (BioTek Synergy HT). The standard curve of a serial dilution of the stock solution was used to estimate the concentration of CORT in each plasma sample (Klukowski 2011). Calculations of intra- and inter-assay variation were 2.1% and 13.8%, respectively (Davies 2013).