2.2.2. Cortisol

Concentrations of salivary cortisol were determined using a double-antibody EIA with a polyclonal rabbit anti-cortisol antibody (R4866). Second antibody-coated plates were prepared by adding 150 μL of anti-rabbit IgG (0.01 mg/mL) to each well of a 96-well microtiter plate (Nunc-Immuno maxisorp, Thermo Fisher Scientific, Roskilde, Denmark), and incubated at RT for 15–24 h. The wells were then emptied and blotted dry, followed by adding 250 μL blocking solution (100 mM phosphate, 150 mM sodium chloride, 1% Tween 20, 0.09% sodium azide, 10% sucrose, pH 7.5) and incubating for 15–24 h at RT. After incubation, all wells were emptied, blotted, and dried at RT in a desiccating cabinet (Sanpla Dry Keeper, Sanplatec Corp., Auto A-3, Japan) with loose desiccant in the bottom. After drying (humidity < 20%), plates were heat-sealed in a foil bag with a 1g desiccant packet and stored at 4 ◦C until use. Neat samples (50 μL) or cortisol standards (50 μL) were added to appropriate wells. Cortisol-horseradish peroxidase (HRP) (25 μL) was immediately added to each well, followed by 25 μL anti-cortisol antibody (except non-specific binding wells) and incubated at RT for 1 h on a plate shaker set to 150 rpm. Plates were then washed four times with wash bu ffer (1:20 dilution, 20× Wash Bu ffer Part No. X007; Arbor Assays, Ann Arbor, MI, USA) and 100 μL of TMB dihydrochloride dissolved in phosphate-citrate bu ffer with sodium perborate (Sigma Aldrich, St. Louis, MO, USA) was added, followed by incubation for 10 min at RT without shaking. The reaction was stopped with 50 μL stop solution (1N HCl) and absorbance was measured at 450 nm by a microplate reader (TECAN Sunrise, Salzburg, Austria). Assay sensitivity based on 90% binding was 0.084 ng/mL. The cortisol EIA was validated for elephant saliva by demonstrating parallelism between serial dilutions of saliva and the cortisol standards (*y* = −10.946*<sup>x</sup>* + 99.705, *R*<sup>2</sup> = 0.996) and significant recovery of cortisol added to low concentration saliva before analysis (*y* = 0.7935*x* + 0.0743, *R*<sup>2</sup> = 0.9987). Samples were analyzed in duplicate; inter-assay and intra assay CVs were 5.48% (n = 4) and 1.46%, respectively.
