| ELISA | |
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A 96-well microtiter plate being used for ELISA. |
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| MeSH | D004797 |
Enzyme-linked immunosorbent assay (ELISA), is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance in a liquid sample or wet sample.
"Wet lab" analytic biochemistry assays involves detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily measured quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well that is needed to keep the reactants contained; as opposed to "dry lab" that can use dry strips - and even if the sample is liquid (eg a measured small drop), the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples.
Heterogenous assays are those assays that needs to separate some component of the analytic reaction mixture, eg by adsorbing some components on to a solid phase which is either physically immobilized eg in ELISA, or spatially separable eg by magnetic or centrifugal or other forms of physical separation, and unwanted components are thrown away (washed or aspirated or centrifuged etc).
In ELISA a liquid sample is added onto a stationary (i.e. not movable or washable) solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (eg color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured by "reading of an optical signal" i.e. quantitative detection of intensity of transmitted light by spectrophotometric method which involves quantitation of transmission of some specific type of light (eg monochromatic i.e. single color) through the liquid (as well as the transparent bottom of the well in the multi-well plate format). The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification process the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantitation - thus the name "Enzyme linked".
The analyte is also called the ligand because it will specifically bind i.e. ligate to a detection reagent and thus ELISA falls under the bigger category of Ligand Binding Assays. The ligand-specific binding reagent is "immobilized" i.e. usually coated and dried onto the transparent bottom and sometimes also side wall of an well (the stationary "solid phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multi-well plate known as the "Elisa Plate". Conventionally, like other forms of immunoassays the specificity of Antigen-Antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively if the analyte itself is an antibody or its target antigen can be used as the binding reagent. But the ELISA format is actually so versatile that as long as any form of specific ligating reagent can be immobilized on the solid phase, and detection reagents will bind specifically and use an enzyme to generate an amplified but predictably quantitative signal, ELISA can perform other forms of ligand binding assays instead of doing only strictly "immuno"assays though the name carried the original "immuno" because of the common use and history of development of this method. The term immunosorbent assay means that in between the washes only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (eg a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase which is part of the plate and thus are not easily reusable.
The ELISA has been used 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 containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the 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 itself be detected by a secondary antibody that 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.
Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques utilize fluorogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable signals. These new reporters can have various advantages including higher sensitivities 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 these assays are largely similar, they are often grouped in the same category as ELISAs.
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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 for determining serum 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 used in toxicology as a rapid presumptive screen for certain classes of drugs.
The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the amount of secondary 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 used for 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." Those that generate weaker signal are "negative."
Doctor Dennis E Bidwell and Alister Voller created the test.
Other uses of ELISA include: 1. detection of mycobacterial antibodies in tuberculosis. 2. detection of rotavirus in feces. 3. detection of hepatitis B markers in the serum. 4. detection of enterotoxin of E. coli in feces.
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 present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.[5]
Because radioactivity poses 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 peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-tetramethylbenzidine), a change in color occurs, which is used as a signal. However, the signal has to be associated with the presence 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]
The steps of "indirect" ELISA follows the mechanism below:-
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-sense limitations, the enzyme can go on producing color indefinitely, but the more primary antibody is present in the donor serum the more secondary 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 serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum 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 serum'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 used 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 serial 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.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay.
A descriptive animation of the application of sandwich ELISA to home pregnancy testing can be found here.
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample the less labeled antigen is retained in the well and the weaker the signal).
It is common that the antigen is not first positioned in the well.
For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody). Competition occurs between the two antibodies for the same antigen. Sera to be tested are added to these wells and incubated at 37 degrees and then washed. If antibodies are present, antigen-antibody reaction occurs. There is no antigen left for the enzyme labelled specific HIV antibodies. These antibodies remain free upon addition and are washed off during washing. Substrate is added but there is no enzyme to act on it, therefore, positive result shows no color change.
A new technique (EP 1 499 894 B1 in EPO Bulletin 25.02.209 N. 2009/09; USPTO 7510687 in USPTO Bulletin 31.03.2009; ZL 03810029.0 in SIPO PRC Bulletin 08.04.2009) uses a solid phase made up of an immunosorbent polystyrene rod with 8-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
The advantages of this technique are as follows:
ELISA tests are used as in in vitro diagnostics in medical laboratories.
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