Determining Cortisol Levels in Human Hair

Materials

• Hair samples collected and stored at -20
• Cayman Chemicals Cortisol Assay Kit (catalog number)
• Clean scissors and tweezers
• Spray bottle of methanol
• Glass slide with 1cm intervals marked

Procedure

Preparation of hair samples

• Stick one side of the hair sample on a plate, use tweezers to grip the hair, use scissors to cut 1 cm hair sample.
• Weigh the hair sample, and add 400uL hexane per mg hair sample. Record the weight of the hair by weighing the empty 2 mL tube, adding the hair and re-weighing it.
• Place 35 mg of hair into a clean tissue homogenizer tube, add 1.5 ml of hexane, and incubate at room temperature for 2 minutes.
• Remove hexane from the tube and dry the hair under a gentle stream of nitrogen.
• Cap the tube and flash freeze the hair sample with liquid nitrogen.
• Use homogenizer to grind the hair sample at 6,800 rpm, 5x20s cycles, pausing for 30 seconds between the cycles.
• Add 1.8 ml of methanol, vortex, and rock overnight on a shaker at room temperature.

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• Collect methanol into a clean test tube. Second tubes (2mL)
• Evaporate methanol under a gentle stream of nitrogen at 37°C.
• Reconstitute in 400 µl of ELISA Buffer (1X) and analyze in the assay

Preparation of the assay reagents

ELISA Buffer (1X) Preparation

• Dilute the contents of one vial of ELISA Buffer Concentrate (10X) (Item No.400060) with 90 ml of ultrapure water. Be certain to rinse the vial to remove any salts that may have precipitated.
• Wash Buffer (1X) Preparation (Item No. 400062)
• Dilute to a total volume of 2L with ultrapure water and add 1 ml of Polysorbate 20.

Preparation of Cortisol ELISA Standard

• Equilibrate a pipette tip by repeatedly filling and expelling the tip with the Cortisol ELISA Standard (Item No. 400364) several times.
• Using the equilibrated pipette tip, transfer 100 µl of the standard into a clean test tube, then dilute with 900 µl ultrapure water → 40 ng/ml.
• Obtain eight clean test tubes and label them #1-8.
• Aliquot 900 µl ELISA Buffer (1X) to tube #1 and 600 µl ELISA Buffer (1X) to tubes #2-8.
• Transfer 100 µl of the bulk standard (40 ng/ml) to tube #1 and mix thoroughly.
• Dilute the standard by removing 400 µl from tube #1 and placing it in tube #2, mix thoroughly.
• Remove 400 µl from tube #2 and place it into tube #3, mix thoroughly. Repeat this process for tubes #4-8.
• Diluted standards should not be stored for more than 24 hours

Preparation of Cortisol-AChE Tracer

• Reconstitute the Cortisol-AChE Tracer (Item No. 10005272) with 6 ml of ELISA Buffer (1X).

Preparation of Cortisol ELISA Monoclonal Antibody

• Reconstitute the Cortisol ELISA Monoclonal Antibody (Item No. 400362) with 6 ml of ELISA Buffer (1X).

Setting up an assay plate

• Draw out a 96 well grid (8 x 12) labelling where you intend to put the blanks, standards,

Addition of the reagents

• Add 100 µl ELISA Buffer (1X) to NSB wells.
• Add 50 µl ELISA Buffer (1X) to blank wells.
• Add 50 µl from standard tube #8 to both of the lowest standard wells. Add 50 µl from standard tube #7 to each of the next standard wells. Continue with this procedure until all the standards are aliquoted. (The same pipette tip should be used to aliquot all the standards. Before pipetting each standard, be sure to equilibrate the pipette tip in that standard.)
• Add 50 µl of sample per well. Each sample should be assayed in duplicate (triplicate recommended).
• Using the multichannel pipet add 50 µl Cortisol-AChE Tracer to each well except the TA and the blank wells.
• Using the multichannel pipet add 50 µl Cortisol ELISA Monoclonal Antibody to each well except the TA, NSB and the blank wells, within 15 min of addition of the tracer.
• Cover each plate with a 96-Well Cover Sheet (Item No. 400012) and incubate overnight at 4°C.
• Reconstitute Ellman’s Reagent (Item No. 400050) immediately before use. Reconstitute 100 dtn vial with 20 ml of ultrapure water. Reconstitute 250 dtn vial with 50 ml of ultrapure water.
• Empty the wells and rinse five times with ~300 µl Wash Buffer (1X).
• Add 200 µl of Ellman’s Reagent to each well.
• Add 5 µl of the reconstituted tracer to the TA wells.
• Cover the plate with the 96-Well Cover Sheet. Optimum development is obtained by using an orbital shaker equipped with a large, flat cover to allow the plate(s) to develop in the dark at room temperature. This assay typically develops (i.e., B0 wells ≥0.6 A.U. (Blk subtracted)) in 90-120 minute

Reading the plate

• Wipe the bottom of the plate with a clean tissue to remove fingerprints, dirt, etc.
• Remove the cover sheet being careful to keep Ellman’s Reagent from splashing on the cover.
• Read the plate at a wavelength between 405 and 420 nm. The absorbance may be checked periodically until the B0 wells have reached a minimum of 0.3 A.U. (Blk subtracted). The plate should be read when the absorbance of the B0 wells is in the range of 0.3-1.0 A.U. (Blk subtracted). If the absorbance of the wells exceeds 1.5 A.U., wash the plate, add fresh Ellman’s Reagent, and let it develop again.