Last updated: 2025-04-22

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Evaluation of Nist et al. 2020

This protocol is different from others in that it should make possible to quantify cortisol in low-mass hair samples (i.e. less than 20 mg). The study conducted by Nist et al. quantifies cortisol in hair samples form neonates, but to our knowledge, it has not been tested with low-mass adult samples.

The increased sensibility of Nist et al. 2020 is provided by two modifications in the traditional protocol:

I tested this protocol by running an ELISA plate with 40 samples from one adult individual. In order to find optimal parameters for my adult samples, I tested different mass, dilution, and the addition (or not) of a spike.

The results suggest that the method proposed by Nist et al. 2020 does not produce reliable results, and does not allow us to quantify cortisol from adult hair. However, it remains a question if using lower-mass samples would result in accurate results (more testing forthcoming).

Among the non-spiked samples, and using a double extraction, we found that a dilution of 250 uL, and using between 20 to 35 mg of hair provides the most consistent results.

This is how data is obtained:

   Plate reader
    |
    |____ optical density (absorbance) 
           |
           |____ Myassays.com (data quality, 
                 |              binding percentages, 
                 |              conc. values (without accounting 
                 |              for weight/dilution/spike)) 
                 |              
                 |____ Data cleaning (ELISA_QC_test#)
                 |     |
                 |     |____ Analysis raw values
                 |
                 |
                 |____ Calculation of final cortisol values (pg/mg)
                       |
                       |____ Analysis of final cortisol values

Find more details in the pages below:

Test 3 Test 4 Test 5 Comments
Data cleaning Test3 Test4 — —
Analysis of raw results Test3 Test4 — Exploration of binding percentages, with the goal of identifying optimal variable values for data generation, so they fall within the ranges measured accurately by the ELISA
Calculation of cortisol values Test3 Test4 — Transformation of raw values obtained from ELISA to cortisol concentration in pg/mL
Models to identify variables affecting binding percentages Test3 — — —