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Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, 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. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed 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. Thus in the case of fluorescence ELISA, when light is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be measured. Look up Elisa in Wiktionary, the free dictionary. ... Wöhler observes the synthesis of urea. ... Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. ... Each antibody binds to a specific antigen; an interaction similar to a lock and key. ... An antigen or immunogen is a molecule that stimulates an immune response. ... In general, diagnosis (plural diagnoses) has two distinct dictionary definitions. ... Phytopathology or Plant Pathology is the science of diagnosing and managing plant diseases. ... For the Jurassic 5 album, see Quality Control (album) In engineering and manufacturing, quality control and quality engineering are involved in developing systems to ensure products or services are designed and produced to meet or exceed customer requirements. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ...


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. For other uses, see Polystyrene (disambiguation). ... Not to be confused with absorption. ... Human glyoxalase I. Two zinc ions that are needed for the enzyme to catalyze its reaction are shown as purple spheres, and an enzyme inhibitor called S-hexylglutathione is shown as a space-filling model, filling the two active sites. ... The primary antibody (in purple) binds to an antigen (in red). ... Laundry detergents are just one of many possible uses for detergents Detergent is a compound, or a mixture of compounds, intended to assist cleaning. ... Look up substrate in Wiktionary, the free dictionary. ... In information theory, a signal is the sequence of states of a communications channel that encodes a message. ... Chromogenic refers to color photographic processes in which a traditional silver image is first formed, and then later replaced with a colored dye image. ...

A 96-well microtiter plate being used for ELISA.
A 96-well microtiter plate being used for ELISA.

Contents

Image File history File linksMetadata Download high resolution version (1984x1488, 715 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): ELISA Microtiter plate Metadata This file contains additional information, probably added from the digital camera or scanner used... Image File history File linksMetadata Download high resolution version (1984x1488, 715 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): ELISA Microtiter plate Metadata This file contains additional information, probably added from the digital camera or scanner used... A 96-well microtiter plate. ...

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.[2] ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs. Blood plasma is the liquid component of blood, in which the blood cells are suspended. ... Randal Tobias, U.S. Global AIDS Coordinator, being publicly tested for HIV/AIDS in Ethiopia in an effort to reduce the stigma of being tested. ... West Nile virus (or WNV) is a virus of the family Flaviviridae; part of the Japanese encephalitis (JE) antigenic complex of viruses, it is found in both tropical and temperate regions. ... A food allergy is an immunologic response to a food protein. ... A glass of cows milk. ... This article is about the legume. ... For other uses, see Walnut (disambiguation). ... For other uses, see Almond (disambiguation). ... Chicken egg (left) and quail eggs (right), the types of egg commonly used as food An egg is a body consisting of an ovum surrounded by layers of membranes and an outer casing of some type, which acts to nourish and protect a developing embryo. ...


The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been 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 negative result.


One method of determining a cut-off point is by comparison with a known standard. For example, if an ELISA test will be used in workplace drug screening, a cut-off concentration (e.g., 50 ng/mL of drug) will be established and a sample will be prepared that contains that concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative."


History

Prior to the development of the EIA/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[3]. An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. ... Radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. ... Radioactive decay is the set of various processes by which unstable atomic nuclei (nuclides) emit subatomic particles. ... Rosalyn Sussman Yalow (born on July 19, 1921) is an American medical physicist, and a co-winner of the 1977 Nobel Prize in Physiology or Medicine for her development of the radioimmunoassay (RIA) technique. ... Solomon Aaron Berson (New York City 22 April 1918 - Atlantic City 11 April 1972) was an American physician whose discoveries, mostly together with Rosalyn Yalow, caused major advances in clinical biochemistry. ...


Because radioactivity poses a 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 certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be 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[4]. 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 Porath in 1966[5] Glutathione Peroxidase 1 A peroxidase (eg. ... In biochemistry, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) or ABTS is chemical compound used to observe the reaction kinetics of specific enzymes. ...


In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA[6][7]


Types

"Indirect" ELISA

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:

  1. Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
  2. The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
  3. A concentrated solution of non-interacting protein, such as Bovine Serum Albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific absorption of other proteins to the plate.
  4. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
  5. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
  6. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
  7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
  8. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will 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 ELISA provides a solution to this problem. A 96-well microtiter plate. ... A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. The assay is first performed with various known concentrations of a substance similar to that being measured. ... Blood plasma is the liquid component of blood, in which the blood cells are suspended. ... Bovine serum albumin, Bovine Albumin, BSA: A serum albumin protein that can be used as a diluent or a blocking agent in numerous applications including ELISAs (Enzyme-Linked Immunosorbent Assay), blots and immunohistochemistry. ... Casein (from Latin caseus cheese) is the most predominant phosphoprotein found in milk and cheese. ... Look up blocking, block in Wiktionary, the free dictionary. ...


ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result 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 is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.


Sandwich ELISA

A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary 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 used to detect sample antigen. The steps are as follows: Image File history File links ELISA-sandwich. ... Image File history File links ELISA-sandwich. ...

  1. Prepare a surface to which a known quantity of capture antibody is bound.
  2. Block any non specific 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 primary antibodies that bind specifically to the antigen.
  6. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
  7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
  8. Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
  9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically 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. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. 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.


Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

  1. Unlabeled antibody is incubated in the presence of its antigen.
  2. These bound antibody/antigen complexes are then added to an antigen coated well.
  3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
  4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
  5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal. The primary antibody (in purple) binds to an antigen (in red). ...


(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.)


ELISA Reverse method & device (ELISA-R m&d)

A newer technique uses an solid phase made up of an immunosorbent polystyrene rod with 4-12 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.


Advantages:

  1. The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
  2. The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
  3. One ogive is left unsensitized to measure the non-specific reactions of the sample;
  4. The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.

See also

An assay is a procedure where the concentration of a component part of a mixture is determined. ... The Enzyme-linked immunosorbent spot (ELISPOT) is a common method for monitoring immune responses in humans and animals. ... An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. ... Radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. ... Secretion assay is a process used in cell biology to identify cells that are secreting a particular protein (usually a cytokine). ...

External links

The University of Arizona (UA or U of A) is a land-grant and space-grant public institution of higher education and research located in Tucson, Arizona, United States. ... Medical Subject Headings (MeSH) is a huge controlled vocabulary (or metadata system) for the purpose of indexing journal articles and books in the life sciences. ...

References

  1. ^ MedLinePlus. "HIV ELISA/western blot." U.S. National Library of Medicine. Last accessed April 16, 2007. http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm
  2. ^ U. S. Food and Drug Administration. "Food Allergen Partnership." Last accessed April 16, 2007. http://www.cfsan.fda.gov/~dms/alrgpart.html
  3. ^ YALOW R, BERSON S (1960). "Immunoassay of endogenous plasma insulin in man". J. Clin. Invest. 39: 1157–75. doi:10.1172/JCI104130. PMID 13846364. 
  4. ^ Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". Clin. Chem. 51 (12): 2415–8. doi:10.1373/clinchem.2005.051532. PMID 16179424. 
  5. ^ Wide L, Porath J. Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies. Biochem Biophys Acta 1966;30:257-260.
  6. ^ Engvall E, Perlman P (1971). "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 871–4. doi:10.1016/0019-2791(71)90454-X. PMID 5135623. 
  7. ^ Van Weemen BK, Schuurs AH (1971). "Immunoassay using antigen-enzyme conjugates.". FEBS Letters 15 (3): 232–6. doi:10.1016/0014-5793(71)80319-8. PMID 11945853. .
A digital object identifier (or DOI) is a standard for persistently identifying a piece of intellectual property on a digital network and associating it with related data, the metadata, in a structured extensible way. ... A digital object identifier (or DOI) is a standard for persistently identifying a piece of intellectual property on a digital network and associating it with related data, the metadata, in a structured extensible way. ... A digital object identifier (or DOI) is a standard for persistently identifying a piece of intellectual property on a digital network and associating it with related data, the metadata, in a structured extensible way. ... A digital object identifier (or DOI) is a standard for persistently identifying a piece of intellectual property on a digital network and associating it with related data, the metadata, in a structured extensible way. ... Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. ... Serology is the scientific study of blood serum. ... The specificity of the bond between antibody and antigen has made it an excellent tool in the detection of substances in a variety of diagnostic techniques, known collectively as diagnostic immunology. ... Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. ... Chromatin immunoprecipitation (ChIP) assay, is an experimental method used in molecular biology. ... Immunodiffusion is a diagnostic test which involves diffusion through a substance such as agar. ... Ouchterlony double immuno diffusion is a simple, rather dated method which is still considered to be the gold standard for detection of ENAs (Extractable Nuclear Antigens). ... Radial immunodiffusion is a immunodiffusion technique used in immunology to detect quantity of antigen by measuring the radius surrounding samples of the antigen, marking the boundary between it and antibody. ... Immunoelectrophoresis (IES) is the electrophoresis of a determined antigen mixture in an agarose gel that allows the separation of different proteins along the gel slide, and then the lateral diffusion in the gel of an immune serum or a monoclonal antibody. ... A laboratory technique used to evaluate the binding of an antibody to its antigen. ... An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. ... The Enzyme Multiplied Immunoassay Technique, or EMIT, is a common method for screening urine and blood for drugs, whether legal or illicit. ... A RAST test (short for radioallergosorbent test) is a blood test used to determine what a person is allergic to. ... Radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. ... Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. ... Agglutination is the clumping of particles. ... Hemagglutination (also haemagglutination) is a more specific form of agglutination that involves red blood cells. ... Coombs test (also known as Coombs test, antiglobulin test or AGT) refers to two clinical blood tests used in [[immunohematology] and immunology. ... Nephelometry is a technique used in immunology to determine levels of IgM, IgG, and IgA.[1] It is performed by shining light on a sample, and measuring the amount of light reflected. ... The complement fixation test is an immunological medical test looking for evidence of infection. ... Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. ... EPITOPE MAPPING This page is a candidate for speedy deletion. ...

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