Enzyme immunoassay kit

Finally, we assessed diurnal rhythm changes over time in cortisol, a biomarker of stress, via saliva concentrations of cortisol determined with enzyme immunoassay kits (obtained from Salimetrics), according to manufacturer instructions. Samples were assayed in duplicate per sample, and the lower limit of sensitivity of the assay was 0.006 μg/dL. Intra- and interassay coefficients of variation of the cortisol enzyme immunoassay were 9.6% and 4.7%, respectively.

All saliva samples were frozen and stored at −80 °C until analysis. For cortisol and DHEA, samples were shipped on dry ice to Dresden Lab Service (Dresden, Germany). After thawing, salivettes were centrifuged at 3000 rpm for 5 min, which resulted in a clear supernatant of low viscosity. Salivary concentrations were measured using commercially available chemiluminescence immunoassays with high sensitivity (IBL International, Hamburg, Germany). The intra- and inter-assay coefficients for cortisol and DHEA were below 8% and 10%, respectively.
For CRP analyses, samples that has been analyzed for cortisol and/or DHEA were shipped on dry ice to the Stress Physiology Investigative Team (SPIT) lab (Iowa State University, Ames, IA, USA) for enzyme immunoassays. Each saliva sample was thawed, centrifuged for 15 min at 3000 rotations per minute, and assayed within 8 h in duplicate using well-established enzyme immunoassay kits (salimetrics). The mean intra-assay coefficient of variation (CV) was 11.6% and mean inter-assay CV was 2%. Samples were reanalyzed if the CV for the duplicate measurements was greater than 10%, based on optical density. To normalize distributions across all assay results, extreme values were winsorized to 2 SDs of the median, and raw values were log-transformed. The number one was added to all log-transformed cortisol values to address negative values.