The enzyme-linked immunosorbent assay ( ELISA ) ( , ) is a test that uses antibodies and discoloration to identify a substance.
ELISA is a popular format of biochemical analytic type "laboratory" using immunoassay solid phase ( EIA ) to detect the presence of a substance, usually antigen, in a liquid sample or a wet sample.
ELISA has been used as a diagnostic tool in the field of medicine and plant pathology, as well as quality control checks in various industries.
The antigen of the sample is attached to the surface. Then, further specific antibodies are applied above the surface so they can bind antigens. These antibodies are related to the enzyme, and, in the final step, the substance containing the enzyme substrate is added. The next reaction generates a detectable signal, most often a color change in the substrate.
Doing ELISA involves at least one antibody with specificity for a particular antigen. Samples with unknown quantities of antigens immobilized in solid support (usually polystyrene microtiter plates) either non-specific (via surface adsorption) or specifically (by capture by other antibodies specific to the same antigen, in ELISA sandwiches). After the antigens are immobilized, detection antibodies are added, forming complexes with antigens. Detection antibodies may be covalently connected with the enzyme, or may be detected by secondary antibodies associated with the enzyme through bioconjugation. Between each step, the plate is usually washed with a mild detergent solution to remove a protein or non-specially bound antibody. After the final washing step, the plate is developed by adding an enzymatic substrate to produce a visible signal, indicating the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding tests instead of rigorous "immuno" tests, although the name carries the original "immuno" due to the general usage and development history of this method. The basic technique requires a binding reagent that can be immobilized in a solid phase along with a detection reagent that will bind specifically and use the enzyme to produce a precisely measured signal. Among the washing, only the ligands and their specific binding pairs remain specially bonded or "immunosorbed" by antigen-antibody interactions into solid phase, whereas non-specific or non-bounding components are drifted. Unlike other wet spectrophotometric test formats in which equally well-reactions (eg quvet) can be reused after washing, the ELISA plate has a reaction product absorbed by the immunosorbed in the solid phase which is part of the plate, so it is not easy to reuse.
Video ELISA
Principles
As an analytical biochemical analysis, ELISA involves the detection of "analyte" (ie a specific substance whose existence is being analyzed quantitatively or qualitatively) in a liquid sample with a method that continues to use liquid reagents during "analysis" (ie a controlled sequence of biochemical reactions that will produce signals that can easily measured and interpreted as a measure of the number of analyte in the sample) that remains liquid and remain in the reaction chamber or necessary to maintain the reactants contained; This is contrary to the "dry laboratories" that can use dry strips - and even if the liquid sample (eg small drop measured), the final detection step in the "dry" analysis involves reading dry strips with methods such as reflectometry and requiring no reaction space for preventing spillover or mixing between samples.
As a heterogeneous test, ELISA separates several components of the analytical reaction mixture by adsorbing certain components into a solid phase that is physically immobilized. In ELISA, the liquid sample is added to the stationary solid phase with special bonding properties and followed by several liquid reagents that are sequentially added, incubated and washed followed by some optical changes (eg the color development by the product of the enzymatic reaction) in the final liquid in the well from which the quantity of analyte is measured. Quantitative "readings" are usually based on the detection of the intensity of light transmitted by spectrophotometry, which involves quantifying the transmission of certain wavelengths of light through the liquid (as well as the transparent bottom of the well in multi-well format plates). The sensitivity of the detection depends on the amplification of the signal during the analytic reaction. Since enzyme reactions are a very well-known amplification process, the signals are generated by enzymes associated with the detection reagent in a fixed proportion to allow for accurate quantification - thus the name "enzyme related".
This analysis is also called ligand because it will specifically bind or bind to the detection reagent, so ELISA belongs to a larger category of ligand binding ligands. The ligand-specific binding reagent is "immobilized", that is, usually coated and dried to the bottom of transparent and sometimes also the well-side wall ("stationary solid"/"solid substrate" here as opposed to dense microparticles/beads can be cleaned), which are usually built as multi-well plates known as "ELISA plates". Conventionally, like other forms of immunoassays, the specificity of the antigen-antibody type is used because it is easy to raise antibodies. specifically against the antigen in bulk as a reagent, or if the analyte itself is an antibody, its target antigen can be used as a binding reagent.
Maps ELISA
History
Prior to the development of ELISA, the only option for immunoassay was radioimmunoassay, a technique that uses radioactive antibodies or antibodies labeled. In radioimmunoassay, radioactivity gives a signal, which indicates whether antigens or specific antibodies are present in the sample. Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.
Because radioactivity poses a potential health threat, safer alternatives are sought. An appropriate alternative to radioimmunoassay will replace nonradioactive signals in lieu of radioactive signals. When enzymes (such as peroxidase radish) react with the appropriate substrate (such as ABTS or TMB), a color change occurs, which is used as a signal. However, signals must be attributed to the presence of antibodies or antigens, which is why the enzymes should be associated with appropriate antibodies. This linking process was developed by Stratis Avrameas and G. B. Pierce. Because it is necessary to remove antibodies or antigens that are not bound by washing, antibodies or antigens must be mounted onto the surface of the container; that is, the immunosorbent must be prepared. The technique for achieving this was published by Wide and Jerker Porath in 1966.
In 1971, Peter Perlmann and Eva Engvall at the University of Stockholm in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands independently published papers that synthesize this knowledge into methods for conducting EIA/ELISA.
Traditional ELISA usually involves reporters and chromogenic substrates that produce a kind of observable color change to indicate the presence of antigens or analytes. Techniques like the newer ELISA use fluorogenic, electrochemiluminescent, and quantitative PCR reporters to make quantitative signals. These new journalists can have many advantages, including higher sensitivity and multiplexing. In technical terms, newer tests of this type are not fully ELISAs, as they are not "connected with enzymes", but are instead associated with some nonenimatic reporters. However, given that the general principles in these tests are very similar, they are often grouped in the same category as ELISA.
In 2012, enzyme-based ultrasensitive and enzyme-based ELISA tests using nanoparticles as chromogenic reporters were able to provide colorless eye signals, from the detection of mere analytic attograms. Blue appears for positive results and red for negative. Note that this detection can only confirm the presence or absence of the analyte instead of the actual concentration.
Type
Direct ELISA
The ELISA steps follow directly the mechanisms below:
- The buffer solution of the antigen to be tested is added to each hole of the microtiter plate, where it is given time to adhere to the plastic through the filling interaction.
- An unreacted protein solution, such as bovine serum albumin or casein, is added to the well (usually a 96-well plates) to cover the plastic surface in a well that remains uncoated by the antigen.
- Primary antibodies with enzyme connected (conjugation) are added, which is binding specifically to test the antigen layer well.
- The substrate for this enzyme is then added. Often, these substrates change color when reacting with enzymes.
- The higher the concentration of primary antibodies present in the serum, the stronger the color changes. Often, the spectrometer is used to provide a quantitative value for the color strength.
Enzyme acts as an amplifier; even if only a few enzyme-related antibodies remain attached, enzyme molecules will produce many signal molecules. In the limitations of common sense, enzymes can continue to produce color indefinitely, but the more antibodies bound, the faster the color will develop. The main disadvantage of direct ELISA is the non-specific antigen immobilization method; when the serum is used as the source of the test antigen, all proteins in the sample may be attached to the microtiter plate well, so the serum analytical concentration in the small serum must compete with other serum proteins when binding to the surface of the well. Sandwich or ELISA does not directly provide a solution to this problem, by using "capturing" antibodies specific to the test antigen to pull it out of the mixture of serum molecules.
ELISA can be run in a qualitative or quantitative format. The qualitative results give a simple positive or negative result (yes or no) to the sample. The cutoff between positive and negative is determined by the analyst and possibly statistically. Two or three times the standard deviation (an inherent error in the test) is often used to differentiate positively from negative samples. In a quantitative ELISA, the optical density (OD) of the sample is compared with the standard curve, which is usually the serial dilution of a known target molecule concentration solution. For example, if the test sample yields OD 1.0, the point on the standard curve giving OD = 1.0 should have the same analytical concentration as the sample.
The use and meaning of the names "direct ELISA" and "indirect ELISA" differ in literature and on the website depending on the context of the experiment. When the presence of the antigen is analyzed, the name "direct ELISA" refers to ELISA using only labeled primary antibodies, and the term "indirect ELISA" refers to ELISA in which the antigen is bound by the primary antibody. which is then detected by labeled secondary antibodies. In the latter case, the ELISA sandwich is distinctly different from the indirect ELISA. When the "main" antibody is interesting, e.g. in the case of immunization analysis, these antibodies are directly detected by secondary antibodies and the term "indirect ELISA" applies to arrangements with two antibodies.
ELISA Sandwich
ELISA "sandwich" is used to detect sample antigen. The steps are:
- A surface is prepared whose quantity of antibody capture is known to be bound.
- All nonspecific binding sites on the surface are blocked.
- Samples containing antigens are applied to plates, and captured by antibodies.
- Plates are washed to remove unbound antigens.
- Specific antibodies are added, and bind to the antigen (hence 'sandwich': the antigen is trapped between two antibodies). These primary antibodies can also be in serum donors to test for reactivity to antigens.
- The enzyme-related secondary enzyme is applied as a detection antibody that also binds specifically to the Fc region of the antibody (nonspecific).
- Plates are washed to remove conjugates of unbound antibodies.
- A chemical is added to be converted by an enzyme into a color or fluorescent or electrochemical signal.
- Absorbance or fluorescence or electrochemical signals (eg, currents) of the wells are measured to determine the presence and quantity of antigens.
The images on the right include the use of conjugated secondary antibodies to the enzyme, although, in a technical sense, this is not necessary if the primary antibody is conjugated to the enzyme (which will be the direct ELISA). However, the use of secondary antibody conjugates avoids the costly process of creating enzyme-related antibodies for any antigen that may want to be detected. By using enzyme-related antibodies that bind to the Fc region of other antibodies, these same enzyme-related antibodies can be used in various situations. Without the first layer of "catching" antibodies, any protein in the sample (including serum proteins) can compete recklessly onto the plate surface, lowering the number of immobilized antigens. The use of purified specific antibodies to attach antigen to the plastic eliminates the need to purify the antigen from the intricate mixture before measurement, simplifying the test, and improving the specificity and sensitivity of the test. ELISA sandwiches used for research often require validation because of the risk of false positive results.
Competitive ELISA
The third use of ELISA is through competitive binding. The steps for ELISA are somewhat different from the first two examples:
- Unlabelled antibodies are incubated in the presence of antigens (samples).
- This bound antibody/antigen complex is then added to the antigen-coated well.
- Plates are washed, so unbound antibodies are removed. (The more antigens in the sample, the more complex Ag-Ab is formed so that there are fewer unbound antibodies available to bind to the antigen in the well, the "competition".)
- Secondary antibodies, specific to primary antibodies, are added. This second antibody is coupled to the enzyme.
- The substrate is added, and the remaining enzyme produces chromogenic or fluorescent signals.
- The reaction is stopped to prevent signal saturation.
Some competitive ELISA kits include enzyme-related antigens rather than enzyme-related antibodies. The labeled antigens compete for primary antibody binding sites with sample antigens (not labeled). The fewer antigens in the sample, the more labeled antigen is retained in the well and the signal gets stronger.
Generally, the antigen is not positioned first in the well.
To detect HIV antibodies, microtiter plate wells are coated with HIV antigens. Two specific antibodies are used, one is conjugated with the enzyme and the other is present in serum (if serum positive for antibody). Cumulative competition occurs between two antibodies for the same antigen, causing a stronger signal to be seen. Sera to be tested were added to this well and incubated at 37 ° C, then washed. If antibodies are present, antigen-antibody reactions occur. No antigen is left for specific HIV antibodies labeled with enzymes. These antibodies remain free at the side and washed during washing. The substrate is added, but there is no enzyme to act on it, so positive results show no discoloration.
Used Ordinary Enzymatic Marker
The following table lists the common enzymatic markers used in the ELISA test.
- OPD ( o -phenylenediamine dihydrochloride) changes to amber to detect HRP (Horseradish Peroxide), which is often used as a conjugate protein.
- TMB (3,3 ', 5,5'-tetramethylbenzidine) is blue when it detects HRP and alters sulfuric acid or yellow phosphate.
- ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt) turns green when it detects HRP
- PNPP ( p -Nitrophenyl Phosphate, Disodium Salt) becomes yellow when it detects alkaline phosphatase.
Apps
Because ELISA can be performed to evaluate the presence of antigens or the presence of antibodies in the sample, it is a useful tool for determining serum antibody concentrations (such as with HIV or West Nile virus tests). He also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs and as serological blood tests for celiac disease. ELISA can also be used in toxicology as a quick alleged screen for certain drug classes.
ELISA is the first widely used screening test for HIV because of its high sensitivity. In ELISA, a person's serum is diluted 400 times and applied to the plate where the HIV antigen is installed. If antibodies to HIV are present in the serum, they may bind these HIV antigens. The dish is then washed to remove all other serum components. A specially prepared "secondary antibodies" - antibodies that bind to other antibodies - are then applied to the plate, followed by another washing. These secondary antibodies are chemically connected first to the enzyme.
Thus, the plate will contain the enzyme in proportion to the number of secondary antibodies bound to the plate. Substrates for enzymes are applied, and catalysis by enzymes causes discoloration or fluorescence. ELISA results are reported as numbers; the most controversial aspect of this test is to determine the "cut-off" point between positive and negative results.
The cut-off point can be determined by comparing it with known standards. If the ELISA test is used for drug screening in the workplace, cut-off concentrations, 50 ng/ml, for example, are established, and samples containing standard concentration analytes will be prepared. It is unknown that it produces a stronger signal than the known sample is "positive." Those who produce weaker signals are "negative".
Dr Dennis E Bidwell and Alister Voller created the ELISA test to detect various types of diseases, such as malaria, Chagas disease, and Johne's disease. ELISA tests are also used as in in vitro diagnostics in medical laboratories. Other ELISA uses include:
- detection of Mycobacterium antibodies in tuberculosis
- detection of rotavirus in feces
- detection of hepatitis B markers in serum
- detection of enterotoxin from E. coli in stool
- detection of HIV antibodies in blood samples
See also
- Immunoscreening
- Lateral flow test
- Magnetic immunoassay
- microtiter plates
- Plaque reduction test of plaque
- The reader plates
- Secretion of secretions
- Agglutination-PCR
Notes and references
External links
Source of the article : Wikipedia
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