ELISA
Encyclopedia
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.
"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 immunoassay
s 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
. 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.
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
or West Nile Virus
). It has also found applications in the food
industry in detecting potential food allergens
such as milk
, peanut
s, walnut
s, almond
s, and eggs
. 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.
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.
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. 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.
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.
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.
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.
(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.
s. 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:
in medical laboratories.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay
Immunoassay
An immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
(EIA) to detect the presence of a substance in a liquid 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
Spectrophotometry
In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
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 immunoassay
Immunoassay
An immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
s the specificity of Antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
-Antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
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
Medical diagnosis
Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...
tool in medicine and plant pathology, as well as a quality-control
Quality control
Quality control, or QC for short, is a process by which entities review the quality of all factors involved in production. This approach places an emphasis on three aspects:...
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
Polystyrene
Polystyrene ) also known as Thermocole, abbreviated following ISO Standard PS, is an aromatic polymer made from the monomer styrene, a liquid hydrocarbon that is manufactured from petroleum by the chemical industry...
microtiter plate
Microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
) either non-specifically (via adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
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
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation
Bioconjugation
Bioconjugation is the process of coupling two biomolecules together in a covalent linkage. Common types of bioconjugation chemistry are amine coupling of lysine amino acid residues , sulfhydryl coupling of cysteine residues , and photochemically initiated free radical reactions, which have broader...
. Between each step, the plate is typically washed with a mild detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
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
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
to produce a visible signal, which indicates the quantity of antigen in the sample.
Traditional ELISA typically involves chromogenic
Chromogenic
Chromogenic refers to color photographic processes in which a traditional silver image is first formed, and then later replaced with a colored dye image.- Description :...
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
Electrochemiluminescence
Electrochemiluminescence or electrogenerated chemiluminescence is a kind of luminescence produced during electrochemical reactions in solutions. In electrogenerated chemiluminescence, electrochemically generated intermediates undergo a highly exergonic reaction to produce an electronically excited...
, and real-time PCR
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...
reporters to create quantifiable signals. These new reporters can have various advantages including higher sensitivities
Sensitivity and specificity
Sensitivity and specificity are statistical measures of the performance of a binary classification test, also known in statistics as classification function. Sensitivity measures the proportion of actual positives which are correctly identified as such Sensitivity and specificity are statistical...
and multiplexing
Multiplex (assay)
A multiplex assay is a type of laboratory procedure that simultaneously measures multiple analytes in a single assay. It is distinguished from procedures that measure one or a few analytes at a time...
. 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.
Applications
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
antibody concentrations (such as with the HIV test
HIV test
HIV tests are used to detect the presence of the human immunodeficiency virus , the virus that causes acquired immunodeficiency syndrome , in serum, saliva, or urine. Such tests may detect antibodies, antigens, or RNA.- Terminology :...
or West Nile Virus
West Nile virus
West Nile virus is a virus of the family Flaviviridae. Part of the Japanese encephalitis antigenic complex of viruses, it is found in both tropical and temperate regions. It mainly infects birds, but is known to infect humans, horses, dogs, cats, bats, chipmunks, skunks, squirrels, domestic...
). It has also found applications in the food
Food
Food is any substance consumed to provide nutritional support for the body. It is usually of plant or animal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals...
industry in detecting potential food allergens
Food allergy
A food allergy is an adverse immune response to a food protein. They are distinct from other adverse responses to food, such as food intolerance, pharmacological reactions, and toxin-mediated reactions....
such as milk
Milk
Milk is a white liquid produced by the mammary glands of mammals. It is the primary source of nutrition for young mammals before they are able to digest other types of food. Early-lactation milk contains colostrum, which carries the mother's antibodies to the baby and can reduce the risk of many...
, peanut
Peanut
The peanut, or groundnut , is a species in the legume or "bean" family , so it is not a nut. The peanut was probably first cultivated in the valleys of Peru. It is an annual herbaceous plant growing tall...
s, walnut
Walnut
Juglans is a plant genus of the family Juglandaceae, the seeds of which are known as walnuts. They are deciduous trees, 10–40 meters tall , with pinnate leaves 200–900 millimetres long , with 5–25 leaflets; the shoots have chambered pith, a character shared with the wingnuts , but not the hickories...
s, almond
Almond
The almond , is a species of tree native to the Middle East and South Asia. Almond is also the name of the edible and widely cultivated seed of this tree...
s, and eggs
Egg (food)
Eggs are laid by females of many different species, including birds, reptiles, amphibians, and fish, and have probably been eaten by mankind for millennia. Bird and reptile eggs consist of a protective eggshell, albumen , and vitellus , contained within various thin membranes...
. 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.
History
Before the development of the ELISA, the only option for conducting an immunoassayImmunoassay
An immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
was radioimmunoassay
Radioimmunoassay
Radioimmunoassay is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies...
, 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
Rosalyn Sussman Yalow
Rosalyn Sussman Yalow was an American medical physicist, and a co-winner of the 1977 Nobel Prize in Physiology or Medicine for development of the radioimmunoassay technique...
and Solomon Berson
Solomon Berson
Solomon Aaron Berson was an American physician and scientist whose discoveries, mostly together with Rosalyn Yalow, caused major advances in clinical biochemistry....
published in 1960.
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
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
) react with appropriate substrates (such as ABTS
ABTS
In biochemistry, 2,2'-azino-bis or ABTS is chemical compound used to observe the reaction kinetics of specific enzymes...
or 3,3’,5,5’-tetramethylbenzidine
3,3’,5,5’-Tetramethylbenzidine
3,3’,5,5’-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays...
), 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. 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
Jerker Porath
Jerker Porath, born 23 October 1921 in Sala, is a Swedish biochemist who has invented several separation methods for biomolecules.Porath studied at Uppsala University and initially did research in organic chemistry under Arne Fredga, where he got his licentiate degree...
in 1966.
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.
"Indirect" ELISA
The steps of "indirect" ELISA follows the mechanism below:-- A buffered solution of the antigen to be tested for is added to each well of a microtiter plateMicrotiter plateA Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
, where it is given time to adhere to the plastic through charge interactions. - A solution of non-reacting protein, such as bovine serum albuminBovine serum albuminBovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard....
or caseinCaseinCasein is the name for a family of related phosphoprotein proteins . These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk....
, is added to blockBlockBlock may refer to:-Administrative subdivisions:* A city block, the smallest area that is surrounded by streets* Block , term used in many South Asian countries-Objects:* A large concrete masonry unit...
any plastic surface in the well that remains uncoated by the antigen. - Next the primary antibody is added, which binds specifically to the test antigen that is coating the well. This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.
- Afterwards, a secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.
- A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in R&D.
- The higher the concentration of the primary antibody that was present in the serum, the stronger the color change. Often a spectrometer is used to give quantitative values for color strength.
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.
Sandwich ELISA
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:- Prepare a surface to which a known quantity of capture antibody is bound.
- Block any nonspecific binding sites on the surface.
- Apply the antigen-containing sample to the plate.
- Wash the plate, so that unbound antigen is removed.
- A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies);
- Apply enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody's Fc region (non-specific).
- Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
- Apply a chemical that is converted by the enzyme into a color or fluorescent or electrochemical signal.
- Measure the absorbency 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, 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
Pregnancy test
A pregnancy test attempts to determine whether or not a woman is pregnant.These markers are found in urine and blood, and pregnancy tests require sampling one of these substances. The first of these markers to be discovered, human chorionic gonadotropin , was discovered in 1930 to be produced by...
can be found here.
Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:- Unlabeled antibody is incubated in the presence of its antigen (Sample).
- These bound antibody/antigen complexes are then added to an antigen-coated well.
- 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.")
- The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
- A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent 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 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.
Multiple and Portable ELISA (M&P ELISA)(ELISA Reverse in published papers)
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 ogiveOgive
An ogive is the roundly tapered end of a two-dimensional or three-dimensional object.-Applied physical science and engineering:In ballistics or aerodynamics, an ogive is a pointed, curved surface mainly used to form the approximately streamlined nose of a bullet or other projectile.The traditional...
s. 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:
- The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and/or different antigens for multi-target assays;
- The sample volume can be increased to improve the test sensitivity in clinical (blood, saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
- One ogive is left unsensitized to measure the non-specific reactions of the sample;
- The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating the development of ready-to-use lab-kits and on-site kits.
Clinical applications
ELISA tests are used as in in vitro diagnosticsIn vitro diagnostics
In vitro diagnostic tests are medical devices intended to perform diagnoses from assays in a test tube, or more generally in a controlled environment outside a living organism...
in medical laboratories.
See also
- AssayAssayAn assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
- Eva EngvallEva EngvallEva Engvall, born 1940, is one of the scientists who invented ELISA in 1971.-Vita:Dr. Engvall earned her Ph.D. from the University of Stockholm in 1975. Her postdoctoral work was done at the University of Helsinki and City of Hope National Medical Center in California, where she was subsequently...
- ELISPOTELISPOTThe Enzyme-linked immunosorbent spot assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983....
- ImmunoassayImmunoassayAn immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
- ImmunoscreeningImmunoscreeningImmunoscreening is a method of biotechnology to detect a polypeptide produced from a cloned gene....
- RadioimmunoassayRadioimmunoassayRadioimmunoassay is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies...
- Secretion assaySecretion assaySecretion assay is a process used in cell biology to identify cells that are secreting a particular protein . It was first developed by Manz et al. in 1995....
- Lateral flow testLateral flow testLateral flow tests also known as Lateral Flow Immunochromatographic Assays are a simple device intended to detect the presence of a target analyte in sample . Most commonly these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use...
- Magnetic immunoassayMagnetic immunoassayMagnetic immunoassay is a novel type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes , radioisotopes or fluorescent moieties . This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of...
- Plaque reduction neutralization testPlaque reduction neutralization testThe Plaque reduction neutralization test is used to quantify the titre of neutralising antibody for a virus.The serum sample or solution of antibody to be tested is diluted and mixed with a viral suspension. This is incubated to allow the antibody to react with the virus. This is poured over a...
External links
- Training video on YouTube: ELISA in Autoimmune Disease Diagnostics
- An animated illustration of an ELISA assay
- The ELISA technique illustrated
- An animated tutorial comparing the direct and indirect ELISA methods
- ELISA Protocol
- "Introduction to ELISA Activity - beginner walkthrough of ELISA used for detecting HIV, including animations at University of ArizonaUniversity of ArizonaThe University of Arizona is a land-grant and space-grant public institution of higher education and research located in Tucson, Arizona, United States. The University of Arizona was the first university in the state of Arizona, founded in 1885...
- Online Tool for ELISA Analysis