ELISA (Enzyme Linked Immuno Sorbent Assay?

blood test: ELISA (Enzyme Linked Immuno Sorbent Assay


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ELISA (Enzyme Linked Immuno Sorbent Assay?
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Reply:The Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.





Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. In simple terms, an unknown amount of antigen in a sample is immobilized on a surface. One then washes a particular antibody over the surface. This antibody is linked to an enzyme that visibly reacts when activated, say by light hitting it in the case of a fluorescent enzyme; the brightness of the fluorescence would then tell you how how much antigen is in your sample.





The Enzyme ImmunoAssay (EIA) is a synonym for the ELISA.





Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.
Reply:The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or antibody (anti-HIV in the screening test for HIV infection) in body fluids or tissue culture supernatents.
Reply:The Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.





Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. In simple terms, an unknown amount of antigen in a sample is immobilized on a surface. One then washes a particular antibody over the surface. This antibody is linked to an enzyme that visibly reacts when activated, say by light hitting it in the case of a fluorescent enzyme; the brightness of the fluorescence would then tell you how how much antigen is in your sample.





The Enzyme ImmunoAssay (EIA) is a synonym for the ELISA


Applications ;


Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test[1] or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs
Reply:Well - it's all in the name.





You take a sample of whatever you are going to test (in your case blood), and you are looking for the abundance of a particular factor (like HIV virus proteins for AIDS tests).





You adsorb (basically "stick down") your sample onto a substrate, and then wash off the excess.


Then you add your primary antibody. This will have been produced in an animal (like a rabbit) to specifically recognise whatever it is you are probing for. The more of your target there is, the more of these antibodies will stick onto the substrate, via your adsorbed target.





You wash again, and then add a second antibody with an enzyme linked to it. This second antibody recognises the *first* antibody (the one that has bound to your target). So again, the more target you have, the more primary antibody, and therefore the more secondary antibody (and enzyme).





Then you wash one last time, and add a substrate that the enzyme will catalyse the breakdown of. Normally, the substrate is colourless, and the product has a strong colour. You leave this for a set amount of time to react, and you look at it.


So - the more enzyme, the more the colour will have developed. This means that, the more of your target had adsorbed, the more the colour will change in the end.





Results are normally obtained by putting your substrate in a machine that detects precisely how much colour has developed (by the absorbance of light at the wavelength absorbed by the coloured product).





By comparing with a set of standards of known amounts of antibody, you can figure out how much original target there was in your sample.





Easy ;-)