Enzyme Immunoassay Of The Steroid Hormone Cortisol Biology Essay
Cortisol is a corticoid produced and secreted by the adrenal secretory organ. It has several maps such as to increase blood sugar and shops it in the liver as animal starch, to modulate fat, protein, purin metamorphosis and to stamp down the immune system. Because of its little size, hydrocortisone is a low antigenic molecule, which means that it is hard to observe utilizing antibodies against it, but this job can be solved associating at it a hapten, which is a big protein bearer molecule that it is easy noticeable by antibodies.In this sort of check the hydrocortisone nowadays in the trial samples competes with a fixed sum of labeled hydrocortisone for sites on an anti-cortisol antibody which was raised in a coney utilizing hydrocortisone linked to bovine serum albumen as the hapten bearer antigen. The protein and hydrocortisone was linked via its C-3 atom.
The labeled hydrocortisone was produced by attaching a peroxidise enzyme to cortisol. The strength of the coloring material was reciprocally relative to the concentration of cortisol nowadays in the samples. In these two practical we have used hydrocortisone linked to bovin serum albumine, which is a hapten, and we have raised anti-cortisol antibodies taken from coney against it.The first practical consists in to bring forth a standardization graph, which is so used to observe the hydrocortisone concentration in trial sample.
The purpose of the experiment was to utilize a set of standard hydrocortisone concentrations to bring forth a standardization graph which was used to find the hydrocortisone oncentration in the trial samples ( 6a.m-24hours ) of a patient.It is clearly known that in a healthy patient the hydrocortisone concentration in blood reaches the highest extremum about around noon, to drop at the lowest value about midnight. In patients who suffer from unwellnesss 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 hold supposed jobs in the patient or eventual errors committed executing the trial.
Method:
We have used two strips of eight microtitre home base Wellss, coated with anti-cortisol antibodies able to adhere the labeled hydrocortisone. Before that, the fictile surface of the Wellss has been covered with milk casein, which prevents an aspecific binding of the hydrocortisone.
To make the standardization graph, we have added 100 µl of labeled hydrocortisone ( peroxidase-cortisol conjugate 0.5µmol/l ) and 50 µl of standard to each well.We incubated for an hr at room temperature, and after rinsing three times with buffer, in order to avoiding aspecific bindings, we added 200 µl of tetra methyl benzidine, the substrate, that responding with the enzyme develops coloring material.After waiting 20 proceedingss we stopped the procedure adding 50 µl of 2M HCl to each well and we read the light optical density at 450 nanometers in a home base reader.The consequences I gained are:Doctor of optometryhydrocortisone concentraion ( nanometer )0.
84201.0420.31.03910.81630.
786100.570300.4731000.247300The graph shows a general tendency like expected.After doing the standardization curve we used the light optical density taken from a sample, we matched the consequences with the standardization curve in order to obtain the concentration.
The values were:Test sample ODCortisol concentration of the trial sample ( nanometer )0.2872356:00 AM0.522610.582280.646230.743140.773110.
89020.5723024 Hs
Discussion:
Using the standardization curve we have matched the sample optical density and we have found the concentration of the sample. The consequences proved that as more unlabeled hydrocortisone was added there was fewer colorss which lead to diminishing OD values. The cortisol concentration was the highest at 6a.m ( 235nM ) and decreased during the twenty-four hours but after there was an addition in cortisol concentration for the 24 hr trial sample ( 30nM ) , likely due to a error in the public presentation.
In fact, add-on of the substrate solution initiates a kinetic reaction, which is terminated by the add-on of the stop solution. Therefore, the add-on of the substrate and the stopping solution should be added in the same sequence to extinguish any clip divergence during reaction. Mistakes committed during the wash stairss may ensue in deceptive consequences.
Enzyme immunochemical assay of the steroid endocrine cortisol – extent of cross reaction
The 2nd practical is an extent of cross reaction. Cross-reactivity is a measuring of antibody response to substances other than the analyte. The purpose of the 2nd experiment is to happen the specificity of the immunochemical assay which was measured utilizing cross responsiveness between hydrocortisone and five different steroids.The five steroids are:Corticosterone ( 11,21 dihydroxy Lipo-Lutin )Cortisone ( 11 dehydro hydrocortisone )17 hydroxy Lipo-LutinPrednisoloneDexamethosone
Method:
Five steroids were supplied for mensurating the cross responsiveness. The steroids were at a concentration of 100µM which was diluted 10-fold to bring forth concentrations of 10µM, 1µM, 100nM, 10nM, 1nM and 0.
1nM. To each of the home base wells 100µL of labelled hydrocortisone were added along with 50µL of the appropriate criterion. The seven criterions along with the negative control were assayed in extra and incubated at 37 & A ; deg ; C. Following incubation the Wellss were washed three times with rinsing buffer and the 200µL of tetra methyl benzidine ( TMB ) was added to each well. Colour was allowed to develop for less than 20minutes and color development was stopped by adding 100µL 2M HCL to each well. Addition of the acid converted the coloring material from bluish to yellow and the light optical density of each sample was read in a home base reader at 450nm.
The consequences were:hydrocortisone concentration ( nM )hydrocortisone ( OD )Concentration ( nanometer )Corticocosterone ( OD )Cortisone ( OD )17 HP ( OD )Prednisolone ( OD )Dexamethasone ( OD )00.23100.4900.
4730.4170.5270.
4830.30.5120.10.4650.4460.4370.
4690.46410.47910.4580.4860.
4280.4450.45030.
422100.4620.4820.
4150.3700.378100.3911000.
5360.4860.3250.2980.
395300.29110000.2590.3930.2590.
2630.3951000.267100000.2390.3600.
1960.1920.4313000.2101000000.1580.2280.1580.
1580.319The per centum cross reaction of the EIA ( Enzyme immunoassay technique ) for finding of hydrocortisone with other corticoids showed that Prednisolone and Dexamethosone had the highest cross responsiveness from the remainder of the five steroid compounds. Cortisone had the least transverse responsiveness. The finding of the intercept was taken at about 0.4 OD as shown in the figure above.The undermentioned compounds were tested for cross responsiveness:compoundsPercentage Cross-Reactivity ( % )Hydrocortisone100corticosterone1.3Cortone Acetate1dexamethosone138Pediapred150hydroxyprogesterone36
Discussion:
Looking both at the graph and the tabular array above, it is possible to see that Prednisolone and Dexamethosone have the highest cross responsiveness, which means that they both response more than the hydrocortisone.
However, Cortisone and Corticosterone with a cross responsiveness of 1 % and 1.3 % , severally, do non hold a large influence on the measuring.In footings of betterments, the cross responsiveness should be calculated at some multiple of the upper mention bound for the cross responding substance, instead than at some arbitrary point on the dose-response curve, and expressed as the evident per centum alteration in the endogenous analyte concentration. This will supply a more clinically utile manner of measuring the likely grade of intervention that would be encountered in everyday pattern.There are, besides, different methods that allow ciphering hydrocortisone in immunochemical assay such as liquid chromatography and mass spectroscopy.But restrictions of these techniques include their high costs which would enable merely specialised research labs utilizing them instead than everyday research labs.
