Our method introduces a novel alternative stress indicator that could be a platform for numerous future studies for measuring cortisol in new non plot and hair matrices. The advantages of this technique are its simplicity and its ability to be used to evaluate stress levels even in slaughtered animals. Establishing a relationship between high cortisol levels and healthy animals or humans using this technique can help with the diagnosis of some diseases.
This method can also be used to study centuries old biological samples such as these old fossils to evaluate stress levels within the sample life span. For cortisol extraction, weigh out 300 plus or minus 10 milligrams of each sample on a digital analytical scale with a 0.0001 accuracy before placing the samples into individual 15 milliliter conical tubes. Add three milliliters of isopropanol to each sample tube and rotate the tubes at 80 rotations per minute for 2.5 minutes to wash out the cortisol and to remove any potential external contamination.
After the last wash, air dry the samples at room temperature for seven days before washing the samples three more times as demonstrated using fresh ultra pure water for each wash. Weigh out 75 plus or minus five milligrams of dried fin or jawbone sample and use a bead beater to finally grind the sample at 50 hertz for 32 minutes. Then, deliver 1.5 milliliters of methanol to each tube of powdered fin or jawbone and place the samples on a tube rotator at 40 rotations per minute for 18 hours at room temperature.
Following cortisol extraction, sediment the sample tissues by centrifugation. And transfer the top one milliliter of the yellowish, organic cortisol containing layer from each sample into individual 1.5 milliliter micro-centrifuged tubes. Dry the cortisol samples at 38 degrees Celsius in a fume hood overnight to evaporate the methanol.
The next morning, add 400 microliters of PBS to each tube and vortex and centrifuge the samples to collect the cortisol. For ELISA Analysis, load 25 microliters of each extracted standard cortisol sample and control in duplicate into the appropriate wells of a 96 well ELISA plate. Load 25 microliters of Assay diluent into two wells to serve as the zero and into each non specific binding well.
Mix 15 microliters of enzyme conjugate with 24 milliliters of Assay diluent and add 200 microliters of conjugate to each well. Mix the plate on a rotator for five minutes at 500 rotations per minute. Followed by a one hour incubation at room temperature.
At the end of the incubation, wash the plate four times with 300 microliters of 1x Wash Buffer per well. Quickly inverting the plate over the sink after each wash to discard the buffer and blotting the plate on a stack of paper towels. After the last wash, add 200 microliters of TMB substrate solution to each well and place the plate onto the rotator for five minutes at 500 rotations per minute.
After mixing, incubate the plate for 25 minutes at room temperature protected from light. At the end of the incubation, add 50 microliters of stop solution to each well. And mix the plate contents on the rotator for three minutes at 500 rotations per minute or until all of the wells have turned from green to yellow.
Then, read the optical density of the samples and use the micro plate software to convert the cortisol levels in each sample to picograms per milligram. In this representative analysis, cortisol levels tended to be higher in fin samples washed with isopropanol than in those washed with water, but no significant differences in fin cortisol levels were observed among sturgeon species. There was no significant interaction between washing solvents and sturgeon species.
Examination of cortisol from H.Huso jawbones to determine whether sturgeon jawbones might be used as an alternative matrix to fins revealed that the washing solvent had no significant effect on the cortisol level measured in H.Huso's sturgeon. Further, the data revealed a high similarity among the fins of three sturgeon species tested and in H.Huso jawbones. It is important to use the bead beater to grind the samples into fine powder and to use an appropriate commercial ELISA Assay Kit for a successful cortisol measurement.
This method can also be used for detecting cortisol in teeth and other hard matrices as cortisol level measurements provide important information about how the environment affects animal biology. The method could be used as a new approach to assessing cortisol levels in human child milk teeth and in pediatric endocrinology, psycho biology, behavioral, archeological, and forensic studies.
