Enzyme linked immunosorbent assay (ELISA)

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Overview
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. 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 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 flourescence ELISA, when light is shone upon the sample, any antigen/antibody complexes will flouresce so that the amount of antigen in the sample can be measured.

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.

The ELISA Reverse method & device (ER m&d) uses an innovative solid phase constituted of an  immunosorbent polystyrene rod with 4-12 protruding ogives; the entire device is suitable to be  introduced in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are easily carried out by immerging the ogives in microwells of standard microplates pre-filled with reagents, sealed and stored until their use. Important advantages: -the rod has its ogives sensitized with different reagents, so to perform the simultaneous detection of different antibodies and different antigens for multi-target assays; - the sample volume can be increased so to increase the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples; - 1 ogive is not sensitized for highlighting the not-specific reactions of the sample; - the use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, so to allow the setting up of ready-to-use lab-kits and on-site kits.

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 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.

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 human 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 more positive than the known sample are called "positive" and those that generate a signal less positive than the known sample are called "negative."

History
Prior to the development of the EIA/ELISA, immunoassays were conducted using radioactively-labeled antigens or antibodies in a technique called 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.

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. 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

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

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.  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 adsorption 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) The plate is washed to remove any unbound detection antibody. After this wash, only the antibody-antigen complexes remain attached to the well.
 * 6) 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.
 * 7) Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
 * 8) Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
 * 9) 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.

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 less-common variant of this technique, called "sandwich" ELISA, is used 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 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 enzyme-linked secondary antibodies which are specific to the antigen.
 * 6) Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
 * 7) Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
 * 8) 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 capture 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.

(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 your sample, the less labeled antigen is retained in the well and the weaker your signal.)

Related chapters

 * Assay
 * Eva Engvall
 * ELISPOT
 * Immunoassay
 * Radioimmunoassay
 * Secretion assay