Salivary Collection Procedures and Cortisol Assay Determinants
Saliva was collected using Salivettes (Sarstedt). Participants were instructed to
accumulate as much saliva in their mouths as possible. They were then told to
remove the cap off the plastic tube and place the swab in their mouth without
using their hands, hold it in their mouth for 1 min, gently moving saliva toward
it without chewing or swallowing it. After 1 min, participants were told to
guide the swab from their mouth back into the plastic tube without touching the
edges of the container with their lips to avoid any potential
cross-contamination of saliva samples. Immediately after testing, the Salivettes
were centrifuged at 3,400 rpm at 1,100 × g using the Hamilton Bell V6500
Vanguard and stored in a freezer at −20°C (−4°F) until analysis.All enzyme immunoassays were carried out on NUNC Maxisorb plates. Cortisol
antibodies (R4866) and matching horseradish peroxidase conjugate were obtained
from C. Munro of the Clinical Endocrinology Laboratory, University of
California, Davis. Steroid standards were taken from Steraloids, Inc. Newport,
Rhode Island. Plates were first coated with 50 μL of antibody stock diluted at
1:8500 in a coating buffer (50 mmol/L bicarbonate buffer pH 9.6), then sealed
and stored for 12–14 hr at 4°C. A 50 μL wash solution (0.15 mol/L NaCl solution
containing 0.5 ml of Tween 20/L) was added to each well to rinse away unbound
antibody. Next, a 50 μL phosphate buffer was added to each well. Plates were
incubated at room temperature for 2 hr before standards, samples, or controls
were added. Two quality control salivary samples, at 30% and 70% binding (the
low and high ends of the sensitivity range of the standard curve), were
prepared. A 50 μL cortisol horseradish peroxidase conjugate was added to each
well, with a 50 μL of standard, sample, or control. Plates remained incubated
for 1 hr after plate loading, then washed with 50 μL wash solution and 100 μL of
a substrate solution of citrate buffer; H