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Cortisol is a corticosteroid produced and secreted by the adrenal gland. It has several functions such as to increase blood sugar and stores it in the liver as glycogen, to regulate fat, protein, purin metabolism and to suppress the immune system. Because of its small size, cortisol is a low antigenic molecule, which means that it is difficult to detect using antibodies against it, but this problem can be solved linking at it a hapten, which is a large protein carrier molecule that it is easy detectable by antibodies.
In this kind of assay the cortisol present in the test samples competes with a fixed amount of labelled cortisol for sites on an anti-cortisol antibody which was raised in a rabbit using cortisol linked to bovine serum albumin as the hapten carrier antigen. The protein and cortisol was linked via its C-3 atom. The labelled cortisol was produced by attaching a peroxidise enzyme to cortisol. The intensity of the colour was inversely proportional to the concentration of cortisol present in the samples. In these two practical we have used cortisol linked to bovin serum albumine, which is a hapten, and we have raised anti-cortisol antibodies taken from rabbit against it.
The first practical consists in to produce a calibration graph, which is then used to detect the cortisol concentration in test sample. The aim of the experiment was to use a set of standard cortisol concentrations to produce a calibration graph which was used to determine the cortisol oncentration in the test samples (6a.m-24hours) of a patient.
It is clearly known that in a healthy patient the cortisol concentration in blood reaches the highest peak approximately around noon, to drop at the lowest value about midnight. In patients who suffer from illnesses and other disturbs these values might be altered.
What I expected from the experiment was to see the sample cortisol concentration following the standard concentration, otherwise I would have supposed problems in the patient or eventual mistakes committed performing the test.
We have used two strips of eight microtitre plate wells, coated with anti-cortisol antibodies able to bind the labelled cortisol. Before that, the plastic surface of the wells has been covered with milk casein, which prevents an aspecific binding of the cortisol.
To create the calibration graph, we have added 100 µl of labelled cortisol (peroxidase-cortisol conjugate 0.5µmol/l) and 50 µl of standard to each well.
We incubated for an hour at room temperature, and after washing three times with buffer, in order to avoiding aspecific bindings, we added 200 µl of tetra methyl benzidine, the substrate, that reacting with the enzyme develops colour.
After waiting 20 minutes we stopped the process adding 50 µl of 2M HCl to each well and we read the light absorbance at 450 nm in a plate reader.
The results I gained are:
cortisol concentraion (nM)
The graph shows a general trend like expected.
After making the calibration curve we used the light absorbance taken from a sample, we matched the results with the calibration curve in order to obtain the concentration. The values were:
Test sample OD
Cortisol concentration of the test sample (nM)
Using the calibration curve we have matched the sample absorbance and we have found the concentration of the sample. The results proved that as more unlabelled cortisol was added there was fewer colours which lead to decreasing OD values. The cortisol concentration was the highest at 6a.m (235nM) and decreased during the day but after there was an increase in cortisol concentration for the 24 hour test sample (30nM), probably due to a mistake in the performance.
In fact, addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Errors committed during the wash steps may result in misleading results.
Enzyme immunoassay of the steroid hormone cortisol - extent of cross reaction
The second practical is an extent of cross reaction. Cross-reactivity is a measurement of antibody response to substances other than the analyte. The aim of the second experiment is to find the specificity of the immunoassay which was measured using cross reactivity between cortisol and five different steroids.
The five steroids are:
Corticosterone (11,21 dihydroxy progesterone)
Cortisone (11 dehydro cortisol)
17 hydroxy progesterone
Five steroids were supplied for measuring the cross reactivity. The steroids were at a concentration of 100µM which was diluted 10-fold to produce concentrations of 10µM, 1µM, 100nM, 10nM, 1nM and 0.1nM. To each of the plate wells 100µL of labelled cortisol were added along with 50µL of the appropriate standard. The seven standards along with the negative control were assayed in duplicate and incubated at 37°C. Following incubation the wells were washed three times with washing buffer and the 200µL of tetra methyl benzidine (TMB) was added to each well. Colour was allowed to develop for less than 20minutes and colour development was stopped by adding 100µL 2M HCL to each well. Addition of the acid converted the colour from blue to yellow and the light absorbance of each sample was read in a plate reader at 450nm.
The results were:
The percentage cross reaction of the EIA (Enzyme immunoassay technique) for determination of cortisol with other corticosteroids showed that Prednisolone and Dexamethosone had the highest cross reactivity from the rest of the five steroid compounds. Cortisone had the least cross reactivity. The determination of the intercept was taken at approximately 0.4 OD as shown in the figure above.
The following compounds were tested for cross reactivity:
Percentage Cross-Reactivity (%)
Looking both at the graph and the table above, it is possible to see that Prednisolone and Dexamethosone have the highest cross reactivity, which means that they both response more than the cortisol. However, Cortisone and Corticosterone with a cross reactivity of 1% and 1.3%, respectively, do not have a big influence on the measurement.
In terms of improvements, the cross reactivity should be calculated at some multiple of the upper reference limit for the cross reacting substance, rather than at some arbitrary point on the dose-response curve, and expressed as the apparent percentage change in the endogenous analyte concentration. This will provide a more clinically useful way of assessing the likely degree of interference that would be encountered in routine practice.
There are, also, different methods that allow calculating cortisol in immunoassay such as liquid chromatography and mass spectrometry.
But limitations of these techniques include their high costs which would enable only specialized laboratories using them rather than routine laboratories.