Salivary cortisol elisa kit

HPA-axis functioning is indexed by cortisol reactivity and recovery following the Face to Face Still Face during the 2-month visit. Assays will be conducted using Salimetrics Salivary Cortisol ELISA Kits. Samples will be run in duplicate with greater than 20% variation between duplicates as the criterion for repeat analysis. Cardiac functioning is operationalized as a 5-min quiet sleep sample of respiratory sinus arrhythmia (RSA) derived from heart rate data using Porges’ method [88 ]. Infant negative emotionality is assessed using two measures at 2 months. Infant irritability is rated on a 7-point scale during the free play task and each phase of the Face to Face Still Face; scores will be averaged across tasks to yield a single measure of irritability [89 (link)]. And at 2 months, mothers complete the negative affectivity subscale of the Infant Behavior Questionnaire-Revised Very Short Form (IBQ-R VSF) [90 (link)].

Salivary cortisol data were available from 320 participants at baseline. The specimen collection and analytic procedures for salivary cortisol measurements have been described previously [36 (link)]. In brief, specimens were collected in Salivette tubes (Sarstedt, Leicester, UK) twice during the same day; in the morning (approximately 2 h after awakening) and in the afternoon (approximately 8 h after awakening). Samples were stored at −20 °C until subsequent cortisol measurements using an enzyme-linked immune sorbent assay (Salivary Cortisol ELISA Kit; Salimetrics, UK). Each ELISA sample was run in duplicate, with morning and afternoon samples from the same participant run on the same 96-well plate, and cortisol concentrations were expressed in micrograms per deciliter (μg/dL). Each plate also contained a cortisol concentration standard curve, as well as interplate controls with known cortisol concentrations, run in duplicates. The correlation coefficients of the standard curves were >0.997 with a median of 0.999 for each plate. The control samples revealed a between-plate coefficient of variation of 7.0% and the median within-plate coefficient of variation for all participant samples was 8.1%.

The procedure of cortisol extraction was adapted from Alderman and Bernier (2009) (link) and Egan et al. (2009) (link). The cortisol group consisted of 48 animals (n = 24 for each temperature). To obtain the samples, the animals were euthanized at EL and ED, weighed, and placed in 2 mL plastic tubes. Each sample corresponded to a pool of four animals. The final number of pooled samples was n = 3 for each temperature and temporal point (Figure 1B). The samples were homogenized in 500 μL of ice-cold 1X PBS buffer using an X1000 Homogenizer Drive (CAT Scientific, Paso Robles, CA, United States), and processed according to the kit instructions (Salivary Cortisol ELISA kit, Salimetrics, Carlsbad, CA, United States). The optical densities (OD) for the duplicate wells were averaged, the non-specific binding OD was subtracted from all wells, and the percent bound was calculated dividing the mean OD of each duplicate (B) by the mean of zero (Bo). The whole-body cortisol concentrations were interpolated in the standard curve using a 4-parameter non-linear regression curve fit (if r > 0.99), normalized with the weight of each sample (each one a pool of four animals) and reported as cortisol concentrations (ng/g body weight) (according to Egan et al., 2009 (link)). Results were expressed as mean ± standard error of the mean (SEM).

For the analyses of the salivary cortisol, the samples were submitted to an extraction process with ether based on the methodology of Zeugswetter et al. [25 (link)]. First, to 100 μL of the sample, 1 mL of diethyl ether was added under slow stirring using an Edmund Buhler KL2 shaker. The agitation continued for 24 h to ensure that all cortisol would be extracted from the cotton. The obtained samples were frozen to make the collection of the organic phase to new tubes easier. After, diethyl ether was removed in a rotary evaporator (Labconco CentriVap) at 45 °C for about 1 h, and 200 μL of phosphate-buffered saline (PBS) was added to each microtube. To concretely determine the concentration of cortisol in saliva, a Salivary Cortisol ELISA Kit (Salimetrics^®, LLC 1-3002) was used according to the manufacturer’s instructions. To obtain the concentrations of cortisol (ng/mL), the results were read at 450 nm, later corrected to 490 nm to discard the interferences, in a PowerWave XS2 (Bio-TeK^® instruments, EUA) microplate reader.

Saliva samples will be centrifuged at 3000 rpm for 15 min at room temperature. The concentration of cortisol in each sample will be calculated using the Salimetrics Salivary Cortisol ELISA kit (Salimetrics, LLC; Carlsbad). The assay sensitivity is 0.193 nmol/L, and the intra- and inter-assay coefficients of variation are 3 and 10%, respectively. The mean cortisol level across the day, the total cortisol level indexed by the area under the curve, and the diurnal cortisol slope will be calculated.
To explore individual trajectories of changes in cortisol levels over time and the complex relationships between different variables, a two-level individual growth curve model using Mplus software will be adopted as cortisol measures at five daily time points are nested within the participants. The method is an appropriate variant of multiple regression modelling for the nested structure of cortisol data.