ELISA
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay(EIA), is a biochemical technique ud mainly in immunology
to detect the prence of an antibody or an antigen in a sample. The ELISA has been ud as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate.
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 itlf be detected by a condary antibody that is linked to an e
nzyme 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.
Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of obrvable color change to indicate the prence of antigen or analyte. Newer ELISA-like techniques utilize fluorogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable signals. The new reporters can have various advantages including higher nsitivities and multiplexing.[1][2] In technical terms, newer assays of this type are not strictly ELISAs, as they are not "enzyme-linked" but are instead linked to some non-enzymatic reporter. However, given that the general principles in the assays are largely similar, they are often grouped in the same category as ELISAs.
Contents
1 Applications
∙ 2 History
∙ 3 Types
o 3.1 "Indirect" ELISA
o 3.2 Sandwich ELISA
o 3.3 Competitive ELISA
o 3.4 Multiple and Portable ELISA (M&P ELISA)(ELISA Rever in published papers)
∙ 4 See also
∙ 5 References
∙ 6 External links
Applications
ELISA results using S-OIV A neuraminida antibody at 1 μg/ml to probe the immunogenic and the corresponding asonal influenza A neuraminida peptides at 50, 10, 2, and 0 ng/ml.
Becau the ELISA can be performed to evaluate either the prence of antigen or the prence of antibody in a sample, it is a uful tool for determining rum antibody concentrations (such as with the HIV test[3] or West Nile Virus). It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.[4] ELISA can also be ud in toxicology as a rapid presumptive screen for certain class of drugs.
The ELISA was the first screening test widely ud for HIV becau of its high nsitivity. In an ELISA, a person's rum is diluted 400-fold and applied to a plate to which HIV antigens are attached. If antibodies to HIV are prent in the rum, they may bind to the HIV antigens. The plate is then washed to remove all other components of the rum. A specially prepared "condary antibody" —an antibody that binds to other antibodies —is then applied to the plate, followed by another wash. This condary antibody is chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the amount of condary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result.
A cut-off point may be determined by comparing it with a known standard. If an ELISA test is ud fo
r drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is established, and a sample that
contains the standard concentration of analyte will be prepared. Unknowns that generate a signal that is stronger than the known sample are "positive." Tho that generate weaker signal are "negative."
Doctor Dennis E Bidwell and Alister Voller created the test. History
Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using
radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal, which indicates whether a specific antigen or antibody is prent in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.[5]
Becau radioactivity pos a potential health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive
signal. When enzymes (such as peroxida) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), a change in color occurs, which is ud as a signal. However, the signal has to be associated with the prence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce.[6] Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container; i.e., the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Jerker Porath in 1966.[7]
In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in The Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA.[8][9]
Types
"Indirect" ELISA
This article may be confusing or unclear to readers. Plea help
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(March 2009)
The steps of "indirect" ELISA follows the mechanism below:- ∙ A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.
∙ A solution of non-reacting protein, such as bovine rum albumin or cain, is added to block any plastic surface in the well that remains uncoated by the antigen.
∙Next the primary antibody is added, which binds specifically to the test antigen that is coating the well. This primary antibody could also be in the rum of a donor to be tested for reactivity towards the antigen.
∙Afterwards, a condary antibody is added, which will bind the primary antibody. This condary antibody often has an enzyme
attached to it, which has a negligible effect on the binding
properties of the antibody.
∙ A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that condary antibody has bound to primary antibody, which
strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical tting, and in R&D.
∙The higher the concentration of the primary antibody that was prent in the rum, the stronger the color change. Often a
spectrometer is ud to give quantitative values for color
strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-n limitations, the enzyme can go on producing color indefinitely, but the more primary antibody is prent in the donor rum the more condary antibody + enzyme will bind, and the faster color will develop. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; when
rum is ud as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in rum must compete with other rum proteins when binding to the well surface. The sandwich or direct ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the rum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation (error inherent in a test) is often ud to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a rial dilution of a known-concentration solution of the target molecule. For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte concentration as your sample.
Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen prent binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked condary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is ud to detect sample antigen. The steps are as follows:
1.Prepare a surface to which a known quantity of capture antibody
is bound.
2.Block any nonspecific binding sites on the surface.
3.Apply the antigen-containing sample to the plate.
4.Wash the plate, so that unbound antigen is removed.
5.Apply enzyme linked primary antibodies as detection antibodies
that also bind specifically to the antigen.
6.Wash the plate, so that the unbound antibody-enzyme conjugates are
removed.
7.Apply a chemical that is converted by the enzyme into a color or
fluorescent or electrochemical signal.
