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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 produces optical density (OD) values | |—> Myassays.com uses ODs and map of plate layout to: 1) Subtract readings for the NSB (non-specific binding) well (0% binding) from all values 2) Normalize values dividing by reading for zero standard (blank, or B0) 3) Fit a 4-Parameter Logistic curve (for a sigmoidal shape) to the standard readings 4) Extrapolate cortisol concentration values from the curve * Obtained values for each replicate separately by treating them as a single sample when providing the plate layout * These values do not control for differences in dilution, sample weight, spike, etc. * Values obtained are in the same unit as the standards provided (pg/ml) | |—> In R I calculate final values using the formula:
o A / B x C / D x E
▪ A = output myassays.com (pg/ml)
▪ B = sample mass (mg)
▪ C = methanol added for extraction (mL)
▪ D = methanol recovered (mL)
▪ E = reconstitution volume (mL)File with complete dataset has the following columns for each single data point: - Plate number - Well (character) - Sample (character) - Category (character) - Weight_mg (numeric) - Buffer_ml (numeric) - Buffer_ml_well (numeric) - Buffer_ml_tube (numeric) - Dilution_sample (numeric) - Spike (numeric: 0 or 1) - Spike_ml_well (numeric) - Spike_ml_tube (numeric) - Dilution_spike (1 if std1, not diluted before adding to well; 4 if std 4 not diluted before adding to well) - Extraction_ratio (numeric) - Comments (character)
Find more details in the pages below: - Background information and experimental design
| Test 3 | Test 4 | Test 5 | Comments | |
|---|---|---|---|---|
| Data cleaning | ||||
| Analysis of raw results | ||||
| 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 | ||||
| Transformation of raw values obtained from ELISA to cortisol concentration in pg/mL | ||||
| Models to identify variables affecting binding percentages |