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Encyclopedia > Enzyme linked immunosorbent assay

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. It uses two antibodies. One antibody is specific to the antigen. The other reacts to antigen-antibody complexes, and is coupled to an enzyme. This second antibody, which accounts for "enzyme-linked" in the test's name, can also cause a chromogenic or fluorogenic substrate to produce a signal. 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 Human Immunodeficiency Virus, 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. Biochemistry is the study of the chemical processes and transformations in living organisms. ... 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 is a substance that stimulates an immune response, especially the production of antibodies. ... Wikipedia does not yet have an article with this exact name. ... The specificity is a statistical measure of how well a binary classification test correctly identifies the negative cases, or those cases that do not meet the condition under study. ... An immune complex is the combination of an epitope with an antibody directed against that epitope. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... In information theory, a signal is the sequence of states of a communications channel that encodes a message. ... 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 (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. ... To meet Wikipedias quality standards, this article or section may require cleanup. ... A glass of cows milk A goat kid feeding on its mothers milk Milk is the nutrient fluid secreted by the mammary glands of female mammals (including monotremes). ... Peanuts is a syndicated daily and Sunday comic strip written and illustrated by Charles M. Schulz, which ran from October 2, 1950, to February 13, 2000, (the day after Schulzs death). ... This article is about the walnut tree. ... This article refers to the plant. ... An egg is an ovum produced by a female animal for reproduction, often prepared as food. ...

Contents

Methods

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

  1. Apply a sample of known antigen to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile.
  2. The plate wells or other surface are then coated with serum samples of unknown antibody concentration, usually diluted in another species' serum. The use of non-human serum prevents non-specific antibodies in the patient's blood from binding to the antigen.
  3. The plate is washed, so that unbound antibody is removed. After this wash, only the antibody-antigen complexes remain attached to the well.
  4. The second antibodies, which will bind to any antigen-antibody complexes, are added to the wells. These second antibodies are coupled to the substrate-modifying enzyme.
  5. Wash the plate, so that excess unbound antibodies are removed.
  6. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal.
  7. View/quantify the result using a spectrophotometer or other optical 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 96-well microtiter plate. ... Blood plasma is the liquid component of blood, in which the blood cells are suspended. ... In biology, a species is one of the basic units of biodiversity. ...


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 antibody is bound.
  2. Apply the antigen-containing sample to the plate.
  3. Wash the plate, so that unbound antigen is removed.
  4. Apply the enzyme-linked antibodies which are also specific to the antigen.
  5. Wash the plate, so that the unbound antibodies are removed.
  6. Apply a chemical which is converted by the enzyme into a fluorescent signal.
  7. View the result: if it fluoresces, then the sample contained antigen.

The image to the right, includes an additional step, the addition of 'detecting antibody', used to avoid the expensive conjugation process that would be necessary to create 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.


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 green). ...


See also

An assay is a procedure where the concentration of a component part of a mixture is determined. ... ELISPOT is an immunological assay based on ELISA (Enzyme-Linked Immunosorbent Assay). ... 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

References

  • Engvall E, Perlman P. "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G", Immunochemistry, 1971 Sep;8(9):871-4 PMID: 5135623
  • Goldsby, R.A., Kindt, T.J., Osborne, B.A. & Kuby, J. Enzyme-Linked Immunosorbent Assay. In: Immunology, 5th ed.(2003), pp. 148-150. W. H. Freeman, New York.

  Results from FactBites:
 
Enzyme-linked immunosorbent assay for brucella antigen detection in human sera. (241 words)
Enzyme-linked immunosorbent assay for brucella antigen detection in human sera.
A sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody coupled to the solid phase was evaluated for the detection of brucella antigen in serum samples.
The standardised assay was performed on 1607 sera from random blood donors, 146 patients with brucellosis, 20 persons in high risk groups and 264 sera from patients with diseases other than brucellosis.
ELISA - Wikipedia, the free encyclopedia (776 words)
The Enzyme-Linked ImmunoSorbent Assay (ELISA for short) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.
It uses two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme.
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 Human Immunodeficiency Virus, HIV test or West Nile Virus) and also for detecting the presence of antigen.
  More results at FactBites »

 
 

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