Cortisol elisa kit

Individual whole-body samples were weighed and homogenized with 5 (w/v) volume per body weight of phosphate buffered saline (pH 7.4) using a rotor homogenizer, according to de Jesus et al. [52 (link)] and Pavlidis et al. [53 (link)]. A total volume of 500 μL of the homogenate was vortexed in a glass tube with 2 mL of diethyl-ether. The diethyl ether layer was allowed to separate in −80 °C and then transferred to a new glass tube, which was placed in a 45 °C water bath for 20 min to allow the diethyl ether to evaporate. Samples were reconstituted in 100 μL of the enzyme immunoassay extraction buffer. Cortisol concentration was quantified with a commercial enzyme immunoassay kit (Cortisol, ELISA kit, Neogen Corporation, Lansing, MI, USA). The cross-reactivity of the antibody with other related steroids is the same as mentioned above.

Cortisol, testosterone, and anti-Müllerian hormone (AMH) in SP were assessed by commercial enzyme immunoassay (EIA) kits. The Cortisol EIA kit (Cortisol ELISA KIT; Neogen^® Corporation, Ayr, UK) presented cross-reactivity with prednisolone (47.4%), cortisone (15.7%), 11-deoxycortisol (15.0%), prednisone (7.83%), corticosterone (4.81%), 6β-hydroxycortisol (1.37%), 17-hydroxyprogesterone (1.36%), deoxycorticosterone (0.94%), progesterone (0.06%), and all other steroids (<0.06%). The Testosterone EIA kit (Testosterone ELISA KIT; Neogen^® Corporation, Ayr, UK) presented cross-reactivity with testosterone glucuronide (16.12%), androstenedione (0.86%), bolandiol (0.86%), testosterone enanthate (0.13%), estriol (0.10%), testosterone benzoate (0.10%), estradiol (0.05%), dehydroepiandrosterone (0.04%), and all other steroids (<0.09%). No significant cross-reactivity or interference between AMH and analogs was observed in the AMH EIA kit (Anti-Mullerian Hormone (AMH); Cloud-Clone^® Corporation, Katy, TX, USA).
Each EIA was biochemically validated for Oryctolagus cuniculus and SP by verifying precision (intra-assay coefficients of variation (CV) from duplicated samples), sensitivity (smallest concentration analyzed), specificity (linearity of dilution), and accuracy (spike-and-recovery test).

A methanol-based extraction protocol was designed based on the method previously described by Tallo-Parra et al. [52 (link)] to extract cortisol from the hair of dairy cows. For each sample, 1.5 mL of pure methanol was added, and the samples were vortexed and then moderately shaken for 18 h at 30 °C (G24 Environmental Incubator Shaker; New Brunswick Scientific Co. Inc., Edison, NJ, USA) for steroid extraction. Following the extraction, samples were centrifuged at 7000× g for 2 min.
Subsequently, 1.2 mL of supernatant was transferred into a new 2 mL Eppendorf tube and then placed in an oven (Heraeus model T6; Kendro Laboratory Products, Langenselbold, Germany) at 38 ºC. Once the methanol was completely evaporated (approximately after 36 h), the dried extracts were reconstituted with 0.15 mL of EIA buffer (1 M phosphate buffered saline) provided by the EIA assay kit (Cortisol ELISA Kit; Neogen^® Corporation, Ayr, UK) and vortexed for 30 s. This dilution was chosen to fall near the 50% bound on the standard curve, the area of greatest assay precision. Then, the samples were immediately stored at −20 °C until analysis.

For FCM analysis, a commercial enzyme immunoassay (EIA) kit (Cortisol ELISA KIT; Neogen^® Corporation, Ayr, U.K.) was used. This EIA kit presents cross-reactivity with prednisolone (47.4%), cortisone (15.7%), 11-deoxycortisol (15.0%), prednisone (7.83%), corticosterone (4.81%), 6β-hydroxycortisol (1.37%), 17-hydroxyprogesterone (1.36%), deoxycorticosterone (0.94%), progesterone (0.06%), and all other steroids (<0.06%).
The EIA was biochemically validated for Panthera leo bleyenberghi and the feces matrix by verifying precision (intra- and inter-assay coefficients of variation (CV) from duplicated samples), sensitivity (smallest steroid concentration analyzed), specificity (linearity of dilution), and accuracy (spike-and-recovery test).
Fecal extracts from P1 and P2 were analyzed at the time of sampling; hence, samples collected after M1 had died were assessed with a different EIA kit. Several randomly chosen samples from P1 and P2 were reanalyzed in the EIA kit used for P3, thus serving as a control to verify the variability between assays through inter-assay precision.

Hair cortisol concentrations were determined using cortisol EIA detection kits (Cortisol ELISA KIT; Neogen Corporation, Ayr, UK) with a sensitivity of 0.32 pg cortisol/mg of hair. The precision within the test was assessed by calculating intra-assay coefficients of variation (CV, where CV = SD/mean × 100) from all duplicate or triplicate samples analyzed. The inter-assay coefficients of variation were calculated from pool samples with markedly different concentrations and analyzed per duplicate in each EIA kit. Linearity under dilution assesses specificity and accuracy, and was calculated by diluting the pool sample at 1:2, 1:5 and 1:8 ratios with the buffer solution included in the EIA kit. The spike-and-recovery test assesses accuracy and was calculated by adding to 50, 100 and 200 μL of pool sample to 200, 100 and 50 μL of pure standard cortisol solution, respectively. Combinations were repeated with three different pure standard cortisol solutions (20, 2, and 0.2 ng/mL) from the initial solution included in the EIA kit. According to the manufacturer, cross-reactivity of the EIA antibody with other steroids is as follows: prednisolone 47.4%, cortisone 15.7%, 11-deoxycortisol 15.0%, prednisone 7.83%, corticosterone 4.81%, 6β-hydroxycortisol 1.37%, 17-hydroxyprogesterone 1.36%, deoxycorticosterone 0.94%. Steroids with a cross-reactivity <0.06% are not presented.

To analyze cortisol levels from blood and skin mucus, a commercial enzyme immunoassay (EIA) kit (Cortisol ELISA KIT; Neogen^® Corporation, Ayr, UK) was used.
Biochemical validation of the EIA was carried out following the methods described by Carbajal and collaborators [50 (link)]. Samples of plasma and skin mucus extracts from several individuals were first pooled and used in each validation test. Precision was evaluated with the intra-assay coefficient of variation (CV), calculated from all duplicated samples analyzed. All samples from each matrix evaluated were analyzed in single assays; therefore, the inter-assay CV was not assessed. The dilution test was applied to assess the specificity of the EIA kit by comparing observed and theoretical values from pools diluted with EIA buffer. To test the assay’s accuracy, the spike-and-recovery test was used, where known volumes of pools were mixed with different volumes and concentrations of pure standard cortisol solution. Finally, we evaluated the sensitivity of the test, given by the smallest amount of cortisol concentration detected.